Xanthine dehydrogenase (XDH) iRNA compositions and methods of use thereof

ABSTRACT

The present invention relates to RNAi agents, e.g., dsRNA agents, targeting the xanthine dehydrogenase (XDH) gene. The invention also relates to methods of using such RNAi agents to inhibit expression of an XDH gene and to methods of treating or preventing an XDH-associated disease in a subject.

RELATED APPLICATIONS

This application is a 35 § U.S.C. 111(a) continuation application which claims the benefit of priority to PCT/US2021/037748, filed on Jun. 17, 2021, which-in turn-claims the benefit of priority to U.S. Provisional Application No. 63/040,587, filed on Jun. 18, 2020, and U.S. Provisional Application No. 63/153,983, filed on Feb. 26, 2021. The entire contents of each of the foregoing applications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Oct. 14, 2021, is named 121301_12903_SL.txt and is 714,599 bytes in size.

BACKGROUND OF THE INVENTION

Reduced renal clearance of uric acid is the result of a number of factors, including defects in uric acid transporter proteins such as SLC2A9, ABCG2, and others, reduced renal excretion due to renal disease, hypothyroidism, volume contraction and volume depletion, acidosis, lead intoxication, and familial nephropathy due to uromodulin deposits; and altered renal clearance due to hyperinsulinemia or insulin resistance in diabetes. Increased synthesis of uric acid is associated with hyperuricemia plus hyperuricosuria; inborn errors of metabolism such as Lesch Nyhan/HPRT deficiency, PRPP synthetase overactivity, and glucose-6-phosphate dehydrogenase deficiency (Von Gierke disease/Glycogen Storage Disease Type Ia); certain situations of high cell turnover (e.g., tumor lysis syndrome); certain situations of high ATP turnover (e.g., glycogen storage diseases, tissue ischemia). Furthermore, conditions such as chronic kidney disease, hypertension, metabolic syndrome, and high fructose intake may result in both increased uric acid synthesis and decreased uric acid clearance.

Chronic elevated serum uric acid (chronic hyperuricemia), typically defined as serum urate levels greater than 6.8 mg/dl (greater than 360 mmol/), the level above which the physiological saturation threshold is exceeded (Mandell, Cleve. Clin. Med. 75:S5-S8, 2008), is associated with a number of diseases. For example, gout is characterized by recurrent attacks of acute inflammatory arthritis that is caused by an inflammatory reaction to uric acid crystals in the joint typically due to insufficient renal clearance of uric acid or excessive uric acid production. Fructose associated gout is associated with variants of transporters expressed in the kidney, intestine, and liver. Chronic elevated uric acid is also associated with non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD), metabolic disorder, cardiovascular disease, type 2 diabetes, and conditions linked to oxidative stress, chronic low grade inflammation, and insulin resistance (Xu et al., J. Hepatol. 62:1412-1419, 2015; Cardoso et al., J. Pediatr. 89:412-418, 2013; Sertoglu et al., Clin. Biochem., 47:383-388, 2014).

Uric acid (also referred to herein as urate) is the final metabolite of endogenous and dietary purine metabolism. Xanthine oxidase (XO) (EC 1.1.3.22) and xanthine dehydrogenase (XDH) (EC 1.17.1.4), which catalyze the oxidation of hypoxanthine to xanthine, and xanthine to uric acid, respectively, are interconvertible forms of the same enzyme. The enzymes are molybdopterin-containing flavoproteins that consist of two identical subunits of approximately 145 kDa. The enzyme from mammalian sources, including man, is synthesized as the dehydrogenase form, but it can be readily converted to the oxidase form by oxidation of sulfhydryl residues or by proteolysis. XDH is primarily expressed in the intestine and the liver, but it is also expressed in other tissues including adipose tissue.

Allopurinol and febuxostat (Uloric®), inhibitors of the XDH form of the enzyme, are commonly used for the treatment of gout. However, their use is contraindicated in patients with co-morbidities common to gout, especially decreased renal function, e.g., due to chronic kidney disease or hepatic impairment. Their use may also be limited in patients with metabolic syndrome, hypertension, dyslipidemia, non-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD), cardiovascular disease, or diabetes (either type 1 or type 2), due to limited organ function from the disease or condition, or due to adverse drug interactions with agents used for the treatment of such conditions.

Currently, treatments for gout do not fully meet patient needs. Therefore, there is a need for additional therapies for subjects that would benefit from reduction in the expression of an XDH gene, such as a subject having an XDH-associated disease or disorder, e.g., gout.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which affect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a gene encoding xanthine dehydrogenase (XDH). The XDH may be within a cell, e.g., a cell within a subject, such as a human subject.

Accordingly, in one aspect the invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of XDH in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 1, 2, or 3 nucleotides from the nucleotide sequence of SEQ ID NO:2. In certain embodiments, the sense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:2. In certain embodiments, the sense strand comprises at least 17 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 17 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:2. In certain embodiments, the sense strand comprises at least 19 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 19 contiguous nucleotides of the nucleotide sequence of SEQ ID NO:2.

In another aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) for inhibiting expression of xanthine dehydrogenase (XDH) in a cell, wherein said dsRNA comprises a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a region of complementarity to an mRNA encoding XDH, and wherein the region of complementarity comprises at least 15 contiguous nucleotides differing by no more than 0, 1, 2, or 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-3 and 6-7. In certain embodiments, the region of complementarity comprises at least 15 contiguous nucleotides of any one of the antisense nucleotide sequences in any one of Tables 2-3 and 6-7. In certain embodiments, the region of complementarity comprises at least 17 contiguous nucleotides of any one of the antisense nucleotide sequences in any one of Tables 2-3 and 6-7. In certain embodiments, the region of complementarity comprises at least 19 contiguous nucleotides of any one of the antisense nucleotide sequences in any one of Tables 2-3 and 6-7. In certain embodiments, the region of complementarity comprises at least 20 contiguous nucleotides of any one of the antisense nucleotide sequences in any one of Tables 2-3 and 6-7. In certain embodiments, the region of complementarity comprises at least 21 contiguous nucleotides of any one of the antisense nucleotide sequences in any one of Tables 2-3 and 6-7.

In one aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) for inhibiting expression of xanthine dehydrogenase (XDH) in a cell, wherein said dsRNA comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of nucleotides 226-269; 1318-1352; 1953-1998; 2351-2394; 2679-2730; 3867-3916; or 4510-4574 of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2.

In one aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) for inhibiting expression of xanthine dehydrogenase (XDH) in a cell, wherein said dsRNA comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of nucleotides 15-45; 121-162; 226-292; 306-338; 379-402; 428-458; 495-521; 873-907; 1318-1344; 1381-1407; 1604-1643; 1700-1723; 1960-1991; 1996-2029; 2044-2067; 2128-2174; 2186-2208; 2289-2345; 2359-2419; 2689-2722; 2699-2721; 2774-2797; 2930-2958; 2987-3064; 3083-3158; 3195-3221; 3248-3293; 3352-3405; 3460-3520; 3524-3571; 3575-3644; 3870-3963; 4143-4176; 4259-4315; 4355-4379; 4395-4467; 4502-4562; 4577-4648; 4658-4752; 4815-4854; 5199-5277; 5284-5310; 5358-5446; or 5677-5713 of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2.

In one aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) for inhibiting expression of xanthine dehydrogenase (XDH) in a cell, wherein said dsRNA comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of nucleotides 123-143; 229-249; 229-251, 230-250; 237-259; 241-263; 271-291; 1320-1342; 1321-1341; 1965-1987; 1971-1993; 2130-2150; 2682-2704; 2689-2711; 2690-2710; 2699-2721; 3880-3900; or 3883-3903 of SEQ ID NO: 1, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2.

In some embodiments, the antisense strand comprises at least 15 contiguous nucleotides differing by nor more than three nucleotides from any one of the antisense strand nucleotide sequences of a duplex selected from the group consisting of AD-1395794.1; AD-1136038.3; AD-1395797.1; AD-1135991.3; AD-1297597.2; AD-1395803.1; AD-1395805.1; AD-1395807.1; AD-1395811.1; AD-1297663.2; AD-1136008.2; AD-1395816.1; AD-1136061.2; AD-1135987.2; AD-1395823.1; AD-1136166.3; and AD-1136169.

In some embodiments, the antisense strand comprises at least 15 contiguous nucleotides differing by no more than three nucleotides from any one of the antisense strand nucleotide sequences of a duplex selected from the group consisting of AD-1136091, AD-1395794, AD-1395805, AD-1136008, AD-1136038, AD-1395823, AD-1395816, AD-1136061, AD-1395811, and AD-1136166.

In some embodiments, the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequences of nucleotides 2699-2721 of SEQ ID NO:1, and the antisense strand comprises at least 15 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2.

In some embodiments, the antisense strand comprises at least 15 contiguous nucleotides differing by no more than three nucleotides from the antisense strand nucleotide sequence of duplex AD-1136091.

In one embodiment, the dsRNA agent comprises at least one modified nucleotide.

In one embodiment, substantially all of the nucleotides of the sense strand; substantially all of the nucleotides of the antisense strand comprise a modification; or substantially all of the nucleotides of the sense strand and substantially all of the nucleotides of the antisense strand comprise a modification.

In one embodiment, all of the nucleotides of the sense strand comprise a modification; all of the nucleotides of the antisense strand comprise a modification; or all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxythimidine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′-phosphate mimic, a nucleotide comprising a 2′-phosphate, e.g., cytidine-2′-phosphate (C2p); guanosine-2′-phosphate (G2p); uridine-2′-phosphate (U2p); adenosine-2′-phosphate (A2p); a thermally destabilizing nucleotide, a glycol modified nucleotide (GNA), and a 2-O-(N-methylacetamide) modified nucleotide; and combinations thereof.

In one embodiment, the modifications on the nucleotides are selected from the group consisting of LNA, glycol nucleic acid (GNA), hexitol nucleic acid (HNA), CeNA, 2′-methoxyethyl, 2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy, 2′-hydroxyl, and glycol; and combinations thereof.

In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a nucleotide comprising a 2′-phosphate, a glycol modified nucleotide (GNA), e.g., Ggn, Cgn, Tgn, or Agn, and, a vinyl-phosphonate nucleotide; and combinations thereof.

In another embodiment, at least one of the modifications on the nucleotides is a thermally destabilizing nucleotide modification.

In one embodiment, the thermally destabilizing nucleotide modification is selected from the group consisting of an abasic modification; a mismatch with the opposing nucleotide in the duplex; and destabilizing sugar modification, a 2′-deoxy modification, an acyclic nucleotide, an unlocked nucleic acid (UNA), and a glycerol nucleic acid (GNA).

The double stranded region may be 19-30 nucleotide pairs in length; 19-25 nucleotide pairs in length; 19-23 nucleotide pairs in length; 23-27 nucleotide pairs in length; or 21-23 nucleotide pairs in length.

In one embodiment, each strand is independently no more than 30 nucleotides in length.

In one embodiment, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

The region of complementarity may be at least 17 nucleotides in length; 19-23 nucleotides in length; or 19 nucleotides in length.

In one embodiment, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides.

In one embodiment, the dsRNA agent further comprises a ligand.

In one embodiment, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA agent.

In one embodiment, the ligand is an N-acetylgalactosamine (GalNAc) derivative.

In one embodiment, the ligand is one or more GalNAc derivatives attached through a monovalent, bivalent, or trivalent branched linker.

In one embodiment, the ligand is

In one embodiment, the dsRNA agent is conjugated to the ligand as shown in the following schematic

and, wherein X is O or S.

In one embodiment, the X is O.

In one embodiment, the dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand, e.g., the antisense strand or the sense strand.

In another embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the 5′-terminus of one strand, e.g., the antisense strand or the sense strand.

In one embodiment, the phosphorothioate or methylphosphonate internucleotide linkage is at the both the 5′- and 3′-terminus of one strand. In one embodiment, the strand is the antisense strand.

In one embodiment, the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

The present invention also provides cells containing any of the dsRNA agents of the invention and pharmaceutical compositions comprising any of the dsRNA agents of the invention.

The pharmaceutical composition of the invention may include dsRNA agent in an unbuffered solution, e.g., saline or water, or the pharmaceutical composition of the invention may include the dsRNA agent is in a buffer solution, e.g., a buffer solution comprising acetate, citrate, prolamine, carbonate, or phosphate or any combination thereof; or phosphate buffered saline (PBS).

In one aspect, the present invention provides a method of inhibiting expression of a xanthine dehydrogenase (XDH) gene in a cell. The method includes contacting the cell with any of the dsRNAs of the invention or any of the pharmaceutical compositions of the invention, thereby inhibiting expression of the XDH gene in the cell.

In one embodiment, the cell is within a subject, e.g., a human subject, e.g., a subject having a xanthine dehydrogenase-(XDH)-associated disease. Such diseases are typically associated with excess uric acid, e.g., serum uric acid.

In one embodiment, the XDH-associated disease is hyperuricemia. In another embodiment, the XDH-associated disease is gout.

In one embodiment, contacting the cell with the dsRNA agent inhibits the expression of XDH by at least 50%, 60%, 70%, 80%, 90%, or 95%.

In one embodiment, inhibiting expression of XDH decreases XDH protein level in serum of the subject by at least 50%, 60%, 70%, 80%, 90%, or 95%.

In one aspect, the present invention provides a method of treating a subject having a disorder that would benefit from reduction in xanthine dehydrogenase (XDH) expression. The method includes administering to the subject a therapeutically effective amount of any of the dsRNAs of the invention or any of the pharmaceutical compositions of the invention, thereby treating the subject having the disorder that would benefit from reduction in XDH expression.

In another aspect, the present invention provides a method of preventing development of a disorder that would benefit from reduction in xanthine dehydrogenase (XDH) expression in a subject having at least one sign or symptom of a disorder who does not yet meet the diagnostic criteria for that disorder. The method includes administering to the subject a prophylactically effective amount of any of the dsRNAs of the invention or any of the pharmaceutical compositions of the invention, thereby preventing the subject progressing to meet the diagnostic criteria of the disorder that would benefit from reduction in XDH expression.

In one embodiment, the disorder is a xanthine dehydrogenase-(XDH)-associated disorder.

In one embodiment, the subject is human.

In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01 mg/kg to about 50 mg/kg.

In one embodiment, the dsRNA agent is administered to the subject subcutaneously.

In one embodiment, the level of XDH in the subject sample(s) is an XDH protein level in a blood or serum sample(s).

In one embodiment, the administration of the agent to the subject causes a decrease in the level of uric acid (e.g., serum uric acid) or a decrease in XDH protein accumulation.

In certain embodiments, the methods of the invention further comprise administering to the subject an additional therapeutic agent.

In certain embodiments, the compositions and methods of the invention are used in combination with other compositions and methods to treat hyperuricemia and/or gout, e.g., allopurinol, oxypurinol, febuxostat, analgesic or anti-inflammatory agents, e.g., NSAIDS.

The present invention also provides kits comprising any of the dsRNAs of the invention or any of the pharmaceutical compositions of the invention, and optionally, instructions for use.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing the uric acid metabolic pathway. XDH is labeled as XO in the schematic.

FIG. 2 depicts the levels of XDH protein in liver samples obtained from Cynomolgus monkeys following treatment with the indicated XDH targeting siRNAs or allopurinol.

FIG. 3 depicts the levels of levels of XDH mRNA in liver samples obtained from Cynomolgus monkeys following treatment with the indicated XDH targeting siRNAs or allopurinol.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a xanthine dehydrogenase (XDH) gene. The gene may be within a cell, e.g., a cell within a subject, such as a human. The use of these iRNAs enables the targeted degradation of mRNAs of the corresponding gene (xanthine dehydrogenase gene) in mammals.

The iRNAs of the invention have been designed to target the human xanthine dehydrogenase gene, including portions of the gene that are conserved in the xanthine dehydrogenase orthologs of other mammalian species. Without intending to be limited by theory, it is believed that a combination or sub-combination of the foregoing properties and the specific target sites or the specific modifications in these iRNAs confer to the iRNAs of the invention improved efficacy, stability, potency, durability, and safety.

Accordingly, the present invention provides methods for treating and preventing a xanthine dehydrogenase-associated disorder, disease, or condition, e.g., a disorder, disease, or condition associated with elevated serum uric acid levels, e.g., hyperuricemia and gout, using iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a xanthine dehydrogenase gene.

The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is up to about 30 nucleotides or less in length, e.g., 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a xanthine dehydrogenase gene. In certain embodiments, the RNAi agents of the disclosure include an RNA strand (the antisense strand) having a region which is about 21-23 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a xanthine dehydrogenase gene.

In certain embodiments, one or both of the strands of the double stranded RNAi agents of the invention is up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA transcript of a xanthine dehydrogenase gene. In some embodiments, such iRNA agents having longer length antisense strands can, for example, include a second RNA strand (the sense strand) of 20-60 nucleotides in length wherein the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

The use of iRNAs of the invention enables the targeted degradation of mRNAs of the corresponding gene (xanthine dehydrogenase gene) in mammals. Using in vitro and in vivo assays, the present inventors have demonstrated that iRNAs targeting a xanthine dehydrogenase gene can potently mediate RNAi, resulting in significant inhibition of expression of a xanthine dehydrogenase gene. Thus, methods and compositions including these iRNAs are useful for treating a subject having a xanthine dehydrogenase-associated disorder, e.g., hyperuricemia and gout.

Accordingly, the present invention provides methods and combination therapies for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of a xanthine dehydrogenase gene, e.g., a xanthine dehydrogenase-associated disease, such as hyperuricemia and gout, using iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an XDH gene.

The present invention also provides methods for preventing at least one symptom in a subject having a disorder that would benefit from inhibiting or reducing the expression of a xanthine dehydrogenase gene, e.g., hyperuricemia and gout.

In certain embodiments, the administration of the dsRNA to the subject causes a decrease in XDH mRNA level, XDH protein level, and the level of serum uric acid.

The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of a xanthine dehydrogenase gene as well as compositions, uses, and methods for treating subjects that would benefit from inhibition or reduction of the expression of a xanthine dehydrogenase gene, e.g., subjects susceptible to or diagnosed with a xanthine dehydrogenase-associated disorder.

I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise. For example, “sense strand or antisense strand” is understood as “sense strand or antisense strand or sense strand and antisense strand.”

The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means±10%. In certain embodiments, about means±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

The term “at least”, “no less than”, or “or more” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 19 nucleotides of a 21 nucleotide nucleic acid molecule” means that 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

As used herein, “no more than” or “or less” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range. As used herein, ranges include both the upper and lower limit.

As used herein, methods of detection can include determination that the amount of analyte present is below the level of detection of the method.

In the event of a conflict between an indicated target site and the nucleotide sequence for a sense or antisense strand, the indicated sequence takes precedence.

In the event of a conflict between a sequence and its indicated site on a transcript or other sequence, the nucleotide sequence recited in the specification takes precedence.

As used herein, the term “xanthine dehydrogenase,” used interchangeably with the term “XDH,” refers to the well-known gene and polypeptide, also known in the art as Xanthine Dehydrogenase/Oxidase, Xanthine Oxidoreductase, XAN1, XDHA, XOR, XO, EC 1.17.1.4, and EC 1.7.2.2.

XDH belongs to the group of molybdenum-containing hydroxylases involved in the oxidative metabolism of purines. The encoded protein has been identified as a moonlighting protein based on its ability to perform mechanistically distinct functions. Xanthine dehydrogenase can be converted to xanthine oxidase by reversible sulfhydryl oxidation or by irreversible proteolytic modification. As used herein, unless clear from context, xanthine dehydrogenase or XDH is understood to include both the xanthine dehydrogenase and xanthine oxidase (“XO” or “XOR”) form of the protein. The protein is expressed predominantly in the intestine and the liver, but is also expressed in adipose tissue. Two transcript variants have been identified for the human isoform of the gene.

The term “XDH” includes human XDH, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession Nos. GI:91823270, GI: 1519244313, and GI:767915203; mouse XDH, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI:575501724; rat XDH, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. GI:8394543; and Macaca fascicularis XDH, transcript variant X1, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession Nos. GI:544482046 and GI:544482048. Additional examples of XDH mRNA sequences are readily available using, e.g., GenBank, UniProt, OMIM, and the Macaca genome project web site.

Exemplary XDH nucleotide sequences may also be found in SEQ ID NOs:1, 3, 5, 7, 9, 11, and 15. SEQ ID NOs:2, 4, 6, 8, 10, 12, and 16 are the antisense sequences of SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 15, respectively.

The term “XDH,” as used herein, also refers to naturally occurring DNA sequence variations of the XDH gene. The term“XDH,” as used herein, also refers to single nucleotide polymorphisms in the XDH gene. Numerous sequence variations within the XDH gene have been identified and may be found at, for example, NCBI dbSNP and UniProt (see, e.g., www.ncbi.nlm.nih.gov/snp?LinkName=gene_snp&from_uid=7498 (which is incorporated herein by reference as of the date of filing this application) which lists over 3000 SNPs in human XDH). In certain embodiments, such naturally occurring variants are included within the scope of the XDH gene sequence.

Further information on XDH can be found, for example, at www.ncbi.nlm.nih.gov/gene/7498 (which is incorporated herein by reference as of the date of filing this application).

Additional examples of XDH mRNA sequences are readily available through publicly available databases, e.g., GenBank, UniProt, OMIM, and the Macaca genome project web site.

The entire contents of each of the foregoing GenBank Accession numbers and the Gene database numbers are incorporated herein by reference as of the date of filing this application.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a xanthine dehydrogenase gene, including mRNA that is a product of RNA processing of a primary transcription product. The target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an XDH gene. In one embodiment, the target sequence is within the protein coding region of XDH.

The target sequence may be from about 19-36 nucleotides in length, e.g., about 19-30 nucleotides in length. For example, the target sequence can be about 19-30 nucleotides, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In some embodiments, the target sequence is about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In still other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 21 to about 23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 1). The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of a xanthine dehydrogenase gene in a cell, e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a single stranded RNA that interacts with a target RNA sequence, e.g., a xanthine dehydrogenase target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA (siRNA) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., a xanthine dehydrogenase (XDH) gene. Accordingly, the term “siRNA” is also used herein to refer to an iRNA as described above.

In certain embodiments, the RNAi agent may be a single-stranded siRNA (ssRNAi) that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded siRNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

In certain embodiments, an “iRNA” for use in the compositions, uses, and methods of the invention is a double stranded RNA and is referred to herein as a “double stranded RNA agent,” “double stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., a xanthine dehydrogenase (XDH) gene. In some embodiments of the invention, a double stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, or modified nucleobase, or any combination thereof. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the invention include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “iRNA” or “RNAi agent” for the purposes of this specification and claims.

In certain embodiments of the instant disclosure, inclusion of a deoxy-nucleotide—which is acknowledged as a naturally occurring form of nucleotide—if present within a RNAi agent can be considered to constitute a modified nucleotide.

The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 19 to 36 base pairs in length, e.g., about 19-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain embodiments, the duplex region is 19-21 base pairs in length, e.g., 21 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 4, 5, 6, 7, 8, 9, 10, 20, 23 or more unpaired nucleotides. In some embodiments, the hairpin loop can be 10 or fewer nucleotides.

In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides.

In certain embodiment, the two strands of double-stranded oligomeric compound can be linked together. The two strands can be linked to each other at both ends, or at one end only. By linking at one end is meant that 5′-end of first strand is linked to the 3′-end of the second strand or 3′-end of first strand is linked to 5′-end of the second strand. When the two strands are linked to each other at both ends, 5′-end of first strand is linked to 3′-end of second strand and 3′-end of first strand is linked to 5′-end of second strand. The two strands can be linked together by an oligonucleotide linker including, but not limited to, (N)n; wherein N is independently a modified or unmodified nucleotide and n is 3-23. In some embodiments, n is 3-10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, wherein N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker can be involved in base-pair interactions with other nucleotides in the linker. The two strands can also be linked together by a non-nucleoside linker, e.g. a linker described herein. It will be appreciated by one of skill in the art that any oligonucleotide chemical modifications or variations describe herein can be used in the oligonucleotide linker.

Hairpin and dumbbell type oligomeric compounds will have a duplex region equal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be equal to or less than 200, 100, or 50, in length. In some embodiments, ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.

The hairpin oligomeric compounds can have a single strand overhang or terminal unpaired region, in some embodiments at the 3′, and in some embodiments on the antisense side of the hairpin. In some embodiments, the overhangs are 1-4, more generally 2-3 nucleotides in length. The hairpin oligomeric compounds that can induce RNA interference are also referred to as “shRNA” herein.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not be, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs. In one embodiment of the RNAi agent, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

In certain embodiments, an iRNA agent of the invention is a dsRNA, each strand of which comprises 19-23 nucleotides, that interacts with a target RNA sequence, e.g., a xanthine dehydrogenase (XDH) gene, to direct cleavage of the target RNA.

In some embodiments, an iRNA of the invention is a dsRNA of 24-30 nucleotides that interacts with a target RNA sequence, e.g., an XDH target mRNA sequence, to direct the cleavage of the target RNA.

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of a double stranded iRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end, or both ends of either an antisense or sense strand of a dsRNA.

In one embodiment of the dsRNA, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

In certain embodiments, the antisense strand of a dsRNA has a 1-10 nucleotides, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, 10-25 nucleotides, 10-20 nucleotides, or 10-15 nucleotides in length. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′ end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′ end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the extended overhang is replaced with a nucleoside thiophosphate. In certain embodiments, the overhang includes a self-complementary portion such that the overhang is capable of forming a hairpin structure that is stable under physiological conditions.

“Blunt” or “blunt end” means that there are no unpaired nucleotides at that end of the double stranded RNA agent, i.e., no nucleotide overhang. A “blunt ended” double stranded RNA agent is double stranded over its entire length, i.e., no nucleotide overhang at either end of the molecule. The RNAi agents of the invention include RNAi agents with no nucleotide overhang at one end (i.e., agents with one overhang and one blunt end) or with no nucleotide overhangs at either end. Most often such a molecule will be double-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., an XDH mRNA.

As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., a xanthine dehydrogenase nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, or 3 nucleotides of the 5′- or 3′-end of the iRNA. In some embodiments, a double stranded RNA agent of the invention includes a nucleotide mismatch in the antisense strand. In some embodiments, the antisense strand of the double stranded RNA agent of the invention includes no more than 4 mismatches with the target mRNA, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand double stranded RNA agent of the invention includes no more than 4 mismatches with the sense strand, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, a double stranded RNA agent of the invention includes a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of the double stranded RNA agent of the invention includes no more than 4 mismatches with the antisense strand, e.g., the sense strand includes 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, 3 nucleotides from the 3′-end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3′-terminal nucleotide of the iRNA agent. In some embodiments, the mismatch(s) is not in the seed region.

Thus, an RNAi agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, a RNAi agent as described herein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent as described herein contains no more than 2 mismatches. In one embodiment, an RNAi agent as described herein contains no more than 1 mismatch. In one embodiment, an RNAi agent as described herein contains 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the mismatch can optionally be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand which is complementary to a region of an XDH gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of an XDH gene. Consideration of the efficacy of RNAi agents with mismatches in inhibiting expression of an XDH gene is important, especially if the particular region of complementarity in an XDH gene is known to have polymorphic sequence variation within the population.

The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.

As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can be, for example, “stringent conditions”, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression, in vitro or in vivo. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogsteen base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between two oligonucleotides or polynucleotides, such as the antisense strand of a double stranded RNA agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding a xanthine dehydrogenase gene). For example, a polynucleotide is complementary to at least a part of a xanthine dehydrogenase mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding a xanthine dehydrogenase gene.

Accordingly, in some embodiments, the antisense polynucleotides disclosed herein are fully complementary to the target XDH sequence.

In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target XDH sequence and comprise a contiguous nucleotide sequence which is at least 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs:1, 3, 5, 7, 9, 11, or 15, or a fragment of any one of SEQ ID NOs:1, 3, 5, 7, 9, 11, or 15, such as about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target XDH sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2-3 and 6-7, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-3 and 6-7, such as about 85%, about 90%, about 95%, or fully complementary.

In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target XDH sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to a fragment of SEQ ID NO:1 selected from the group of nucleotides 226-269; 1318-1352; 1953-1998; 2351-2394; 2679-2730; 3867-3916; or 4510-4574 of SEQ ID NO: 1, such as about 85%, about 90%, about 95%, or fully complementary.

In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target XDH sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to a fragment of SEQ ID NO:1 selected from the group of nucleotides 15-45; 121-162; 226-292; 306-338; 379-402; 428-458; 495-521; 873-907; 1318-1344; 1381-1407; 1604-1643; 1700-1723; 1960-1991; 1996-2029; 2044-2067; 2128-2174; 2186-2208; 2289-2345; 2359-2419; 2689-2722; 2699-2721; 2774-2797; 2930-2958; 2987-3064; 3083-3158; 3195-3221; 3248-3293; 3352-3405; 3460-3520; 3524-3571; 3575-3644; 3870-3963; 4143-4176; 4259-4315; 4355-4379; 4395-4467; 4502-4562; 4577-4648; 4658-4752; 4815-4854; 5199-5277; 5284-5310; 5358-5446; or 5677-5713 of SEQ ID NO: 1, such as about 85%, about 90%, about 95%, or fully complementary.

In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target XDH sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to a fragment of SEQ ID NO:1 selected from the group of nucleotides 123-143; 229-249; 229-251, 230-250; 237-259; 241-263; 271-291; 1320-1342; 1321-1341; 1965-1987; 1971-1993; 2130-2150; 2682-2704; 2689-2711; 2690-2710; 2699-2721; 3880-3900; or 3883-3903 of SEQ ID NO: 1, such as about 85%, about 90%, about 95%, or fully complementary.

In some embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target XDH sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to a fragment of SEQ ID NO:1, e.g., nucleotides 2699-2721 of SEQ ID NO:1, such as about 85%, about 90%, about 95%, or fully complementary.

In one embodiment, an RNAi agent of the disclosure includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is the same as a target XDH sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, or 16, or a fragment of any one of SEQ ID NOs:2, 4, 6, 8, 10, 12, or 16, such as about 85%, about 90%, about 95%, or fully complementary.

In some embodiments, an RNAi agent of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target xanthine dehydrogenase sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the antisense strand nucleotide sequences in any one of Tables 2-3 and 6-7, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 2-3 and 6-7, such as about 85%, about 90%, about 95%, or fully complementary.

In some embodiments, the double-stranded region of a double-stranded iRNA agent is equal to or at least, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.

In some embodiments, the antisense strand of a double-stranded iRNA agent is equal to or at least 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, the sense strand of a double-stranded iRNA agent is equal to or at least 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 18 to 30 nucleotides in length.

In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 19 to 25 nucleotides in length.

In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each 21 to 23 nucleotides in length.

In one embodiment, the sense strand of the iRNA agent is 21-nucleotides in length, and the antisense strand is 23-nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs having a 2-nucleotide long single stranded overhangs at the 3′-end.

In some embodiments, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide or a modified nucleotide. In addition, an “iRNA” may include ribonucleotides with chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications, as used in an iRNA molecule, are encompassed by “iRNA” for the purposes of this specification and claims.

In certain embodiments of the instant disclosure, inclusion of a deoxy-nucleotide if present within an RNAi agent can be considered to constitute a modified nucleotide.

In one embodiment, at least partial suppression of the expression of an XDH gene, is assessed by a reduction of the amount of XDH mRNA which can be isolated from or detected in a first cell or group of cells in which an XDH gene is transcribed and which has or have been treated such that the expression of an XDH gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition may be expressed in terms of:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

The phrase “contacting a cell with an iRNA,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an iRNA includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the iRNA may be put into physical contact with the cell by the individual performing the method, or alternatively, the iRNA may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating the cell with the iRNA. Contacting a cell in vivo may be done, for example, by injecting the iRNA into or near the tissue where the cell is located, or by injecting the iRNA into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the iRNA may contain or be coupled to a ligand, e.g., GalNAc, that directs the iRNA to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an iRNA and subsequently transplanted into a subject.

In certain embodiments, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusion or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or are known in the art.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an iRNA or a plasmid from which an iRNA is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a rabbit, a sheep, a hamster, a guinea pig, a dog, a rat, or a mouse), or a bird that expresses the target gene, either endogenously or heterologously. In an embodiment, the subject is a human, such as a human being treated or assessed for a disease or disorder that would benefit from reduction in XDH expression; a human at risk for a disease or disorder that would benefit from reduction in XDH expression; a human having a disease or disorder that would benefit from reduction in XDH expression; or human being treated for a disease or disorder that would benefit from reduction in XDH expression as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject.

As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result, such as reducing at least one sign or symptom of an XDH-associated disorder in a subject. Treatment also includes a reduction of one or more sign or symptoms associated with unwanted XDH expression; diminishing the extent of unwanted XDH activation or stabilization; amelioration or palliation of unwanted XDH activation or stabilization. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.

The term “lower” in the context of the level of XDH in a subject or a disease marker or symptom refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, a decrease is at least 20%. In certain embodiments, the decrease is at least 50% in a disease marker, e.g., protein or gene expression level. “Lower” in the context of the level of XDH in a subject is a decrease to a level accepted as within the range of normal for an individual without such disorder. In certain embodiments, the expression of the target is normalized, i.e., decreased towards or to a level accepted as within the range of normal for an individual without such disorder, e.g., normalization of serum uric acid level. As used here, “lower” in a subject can refer to lowering of gene expression or protein production in a cell in a subject does not require lowering of expression in all cells or tissues of a subject. For example, as used herein, lowering in a subject can include lowering of gene expression or protein production in the liver of a subject.

The term “lower” can also be used in association with normalizing a symptom of a disease or condition, i.e. decreasing the difference between a level in a subject suffering from an XDH-associated disease towards or to a level in a normal subject not suffering from an XDH-associated disease.

As used herein, if a disease is associated with an elevated value for a symptom, “normal” is considered to be the upper limit of normal. If a disease is associated with a decreased value for a symptom, “normal” is considered to be the lower limit of normal.

As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, that would benefit from a reduction in expression of an XDH gene or production of XDH protein, refers to preventing a subject who has at least one sign or symptom of a disease from developing further signs and symptoms thereby meeting the diagnostic criteria for that disease. In certain embodiments, prevention includes delayed progression to meeting the diagnostic criteria of the disease by days, weeks, months or years as compared to what would be predicted by natural history studies or the typical progression of the disease.

As used herein, the terms “xanthine dehydrogenase-associated disease” or “XDH-associated disease,” include a disease, disorder or condition that would benefit from a decrease in XDH gene expression, replication, or protein activity. Such disorders are caused by, or associated with elevated serum uric acid levels, such as hyperuricemia, including asymptomatic hyperuricemia.

“Hyperuricemia” is an elevated uric acid level in the blood. The normal upper limit is 6.8 mg/dL, and anything over 7 mg/dL is considered saturated, and symptoms can occur. Additional indicators of hyperuricemia include, for example, accelerated purine degradation, in high cell turnover states (hemolysis, rhabdomyolysis, and tumor lysis) and decreased excretion (renal insufficiency and metabolic acidosis). Methods to detect and monitor uric acid in serum or other subject samples are known in the art. Uric acid levels can be detected, for example using carbonate-phosphotungstate method, spectrophotometric uricase method, or chromatography methods such as HPLC or LCMS.

Hyperuricemia is associated with a number of diseases and conditions, including, but not limited to, gout NAFLD, NASH, metabolic disorder, insulin resistance, cardiovascular disease, hypertension, type 2 diabetes, and conditions linked to oxidative stress e.g., chronic low grade inflammation; or other XDH-associated disease. Thus, the compositions and methods of the invention which lower serum urine acid levels, e.g., lower serum uric acid levels to about to 6.8 mg/dl or less (the level of solubility of uric acid in serum) are also useful to treat subjects having gout, NAFLD, NASH, metabolic disorder, insulin resistance, cardiovascular disease, hypertension, type 2 diabetes, or conditions linked to oxidative, even in the absence of symptoms

“Gout” is a disorder that allows for the accumulation of uric acid in the blood and tissues. This leads to the precipitation of urate monohydrate crystals within a joint. When tissues are saturated with urate, crystals will precipitate. Precipitation is enhanced in acidic environments and cold environments, leading to increased precipitation in peripheral joints, such as the great toe. Gout has a male predominance in a 4:1 ratio of men to women. Uric acid levels can be elevated ten to 15 years before clinical manifestations of gout.

NAFLD is associated with hyperuricemia (Xu et al., J. Hepatol. 62:1412-1419, 2015). The diagnosis of “nonalcoholic fatty liver disease” (“NAFLD”) requires that (a) there is evidence of hepatic steatosis, either by imaging or by histology and (b) there are no causes for secondary hepatic fat accumulation such as significant alcohol consumption, use of steatogenic medication or hereditary disorders. In the majority of patients, NAFLD is associated with metabolic risk factors such as obesity, diabetes mellitus, and dyslipidemia. NAFLD is histologically further categorized into “nonalcoholic fatty liver” (“NAFL”) and “nonalcoholic steatohepatitis” (“NASH”). “NAFL” is defined as the presence of hepatic steatosis with no evidence of hepatocellular injury in the form of ballooning of the hepatocytes. “NASH” is defined as the presence of hepatic steatosis and inflammation with hepatocyte injury (ballooning) with or without fibrosis (Chalasani et al., Hepatol. 55:2005-2023, 2012). It is generally agreed that patients with simple steatosis have very slow, if any, histological progression, while patients with NASH can exhibit histological progression to cirrhotic-stage disease. The long term outcomes of patients with NAFLD and NASH have been reported in several studies. Their findings can be summarized as follows; (a) patients with NAFLD have increased overall mortality compared to matched control populations, (b) the most common cause of death in patients with NAFLD, NAFL, and NASH is cardiovascular disease, and (c) patients with NASH (but not NAFL) have an increased liver-related mortality rate. In a mouse model of NAFLD, treatment with allopurinol both prevented the development of hepatic steatosis, but also significantly ameliorated established hepatic steatosis in mice (Xu et al., J. Hepatol. 62:1412-1419, 2015).

Cardiovascular disease has been associated with hyperuricemia. Allopurinol has been demonstrated to be effective in the treatment of cardiovascular disease in animal models and humans including myocardial infarction, ischemia-reperfusion injury, hypoxia, ischemic heart disease, heart failure, hypercholesterolemia, and hypertension (Pacher et al., Pharma. Rev. 58:87-114, 2006). As discussed above, treatment with allopurinol is contraindicated in a number of populations, especially those with compromised renal function.

Metabolic syndrome, insulin resistance, and type 2 diabetes are associated with hyperuricemia (Cardoso et al., J. Pediatr. 89:412-418, 2013).

“Metabolic syndrome” is characterized by a cluster of conditions defined as at least three of the five following metabolic risk factors which include:large waistline (≥35 inches for women or ≥40 inches for men); high triglyceride level (≥150 mg/dl); low HDL cholesterol (≤50 mg/dl for women or ≤40 mg/dl for men); elevated blood pressure (≥130/85) or on medicine to treat high blood pressure; and high fasting blood sugar (≥100 mg/dl) or being in medicine to treat high blood sugar.

“Insulin resistance” is characterized by the presence of at least one of: a fasting blood glucose level of 100-125 mg/dL taken at two different times; or an oral glucose tolerance test with a result of a glucose level of 140-199 mg/dL at 2 hours after glucose consumption.

“Type 2 diabetes” is characterized by at least one of: a fasting blood glucose level ≥126 mg/dL taken at two different times; ahemoglobin A1c (A1C) test with a result of ≥6.5% or higher; or an oral glucose tolerance test with a result of a glucose level ≥200 mg/dL at 2 hours after glucose consumption.

Metabolic syndrome, insulin resistance, and type 2 diabetes are often associated with decreased renal function or the potential for decreased renal function.

Further details regarding signs and symptoms of the various diseases or conditions are provided herein and are well known in the art.

In one embodiment of the invention, the XDH-associated disease is hyperuricemia.

In another embodiment of the invention, the XDH-associate disease is gout.

In certain embodiments, an XDH-associated disease is NASH or NAFLD.

“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having an XDH-associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating, or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated. In certain embodiments, treatment with the iRNAs of the invention will result in serum uric acid levels at 2-6.8 mg/dl, such as, 2-6 mg/dl in subjects. Maintenance of such uric acid levels will treat or prevent XDH-associated diseases.

“Prophylactically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having at least one sign or symptom of an XDH-associated disorder, is sufficient to prevent or delay the subject's progression to meeting the full diagnostic criteria of the disease. Prevention of the disease includes slowing the course of progression to full blown disease. The “prophylactically effective amount” may vary depending on the RNAi agent, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylactically effective amount” also includes an amount of an RNAi agent that produces some desired effect at a reasonable benefit/risk ratio applicable to any treatment. The iRNA employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Such carriers are known in the art. Pharmaceutically acceptable carriers include carriers for administration by injection.

The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs, or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). In some embodiments, a “sample derived from a subject” refers to urine obtained from the subject. A “sample derived from a subject” can refer to blood or blood derived serum or plasma from the subject.

II. iRNAs of the Invention

The present invention provides iRNAs which inhibit the expression of a xanthine dehydrogenase gene. In some embodiments, the iRNA includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an XDH gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human susceptible to developing a xanthine dehydrogenase-associated disorder. The dsRNAi agent includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an XDH gene. The region of complementarity is about 19-30 nucleotides in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, or 19 nucleotides in length). Upon contact with a cell expressing the XDH gene, the iRNA inhibits the expression of the XDH gene (e.g., a human, a primate, a non-primate, or a rat XDH gene) by at least about 50% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, western blotting or flow cytometric techniques. In some embodiments, inhibition of expression is determined by the qPCR method provided in the examples herein with the siRNA at, e.g., a 10 nM concentration, in an appropriate organism cell or cell line provided therein. In some embodiments, inhibition of expression in vivo is determined by knockdown of the human gene in a rodent expressing the human gene, e.g., a mouse or an AAV-infected mouse expressing the human target gene, e.g., when administered as single dose, e.g., at 3 mg/kg at the nadir of RNA expression.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of an XDH gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

Generally, the duplex structure is 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain embodiments, the duplex structure is 18 to 25 base pairs in length, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24 or 24-25 base pairs in length, for example, 19-21 basepairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

Similarly, the region of complementarity to the target sequence is 15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, for example 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

In some embodiments, the duplex structure is 19 to 30 base pairs in length. Similarly, the region of complementarity to the target sequence is 19 to 30 nucleotides in length.

In some embodiments, the dsRNA is about 19 to about 23 nucleotides in length, or about 25 to about 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well-known in the art that dsRNAs longer than about 21-23 nucleotides in length may serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 19 to about 30 base pairs, e.g., about 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target xanthine dehydrogenase gene expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1-4, 2-4, 1-3, 2-3, 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end, or both ends of an antisense or sense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art. Double stranded RNAi compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Similarly, single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.

Regardless of the method of synthesis, the siRNA preparation can be prepared in a solution (e.g., an aqueous or organic solution) that is appropriate for formulation. For example, the siRNA preparation can be precipitated and redissolved in pure double-distilled water, and lyophilized. The dried siRNA can then be resuspended in a solution appropriate for the intended formulation process.

In an aspect, a dsRNA of the invention includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of sequences provided in any one of Tables 2-3 and 6-7, and the corresponding antisense strand of the sense strand is selected from the group of sequences of any one of Tables 2-3 and 6-7. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of a xanthine dehydrogenase gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in any one of Tables 2-3 and 6-7, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in any one of Tables 2-3 and 6-7.

In certain embodiments, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In other embodiments, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

It will be understood that, although the sequences in Tables 2 and 6 are not described as modified or conjugated sequences, the RNA of the iRNA of the invention e.g., a dsRNA of the invention, may comprise any one of the sequences set forth in any one of Tables 2-3 and 6-7 that is un-modified, un-conjugated, or modified or conjugated differently than described therein. In other words, the invention encompasses dsRNA of Tables 2-3 and 6-7 which are un-modified, un-conjugated, modified, or conjugated, as described herein.

The skilled person is well aware that dsRNAs having a duplex structure of about 20 to 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., EMBO 2001, 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in any one of Tables 2-3 and 6-7, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes having any one of the sequences in any one of Tables 2-3 and 6-7 minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 19, 20, or more contiguous nucleotides derived from any one of the sequences of any one of Tables 2-3 and 6-7, and differing in their ability to inhibit the expression of a xanthine dehydrogenase gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present invention.

In addition, the RNAs provided in Tables 2-3 and 6-7 identify a site(s) in a xanthine dehydrogenase transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 19 contiguous nucleotides from any one of the sequences provided in any one of Tables 2-3 and 6-7 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a xanthine dehydrogenase gene.

An RNAi agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, an RNAi agent as described herein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent as described herein contains no more than 2 mismatches. In one embodiment, an RNAi agent as described herein contains no more than 1 mismatch. In one embodiment, an RNAi agent as described herein contains 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the mismatch can optionally be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand which is complementary to a region of an XDH gene generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of an XDH gene. Consideration of the efficacy of RNAi agents with mismatches in inhibiting expression of an XDH gene is important, especially if the particular region of complementarity in an XDH gene is known to have polymorphic sequence variation within the population.

III. Modified iRNAs of the Invention

In certain embodiments, the RNA of the iRNA of the invention e.g., a dsRNA, is un-modified, and does not comprise, e.g., chemical modifications or conjugations known in the art and described herein. In other embodiments, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the invention, substantially all of the nucleotides of an iRNA of the invention are modified. In other embodiments of the invention, all of the nucleotides of an iRNA or substantially all of the nucleotides of an iRNA are modified, i.e., not more than 5, 4, 3, 2, or 1 unmodified nucleotides are present in a strand of the iRNA.

The nucleic acids featured in the invention can be synthesized or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. In some embodiments of the invention, the dsRNA agents of the invention are in a free acid form. In other embodiments of the invention, the dsRNA agents of the invention are in a salt form. In one embodiment, the dsRNA agents of the invention are in a sodium salt form. In certain embodiments, when the dsRNA agents of the invention are in the sodium salt form, sodium ions are present in the agent as counterions for substantially all of the phosphodiester or phosphorothiotate groups present in the agent. Agents in which substantially all of the phosphodiester or phosphorothioate linkages have a sodium counterion include not more than 5, 4, 3, 2, or 1 phosphodiester or phosphorothioate linkages without a sodium counterion. In some embodiments, when the dsRNA agents of the invention are in the sodium salt form, sodium ions are present in the agent as counterions for all of the phosphodiester or phosphorothiotate groups present in the agent.

Representative U.S. Patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6, 239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH₂ component parts.

Representative U.S. Patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

Suitable RNA mimetics are contemplated for use in iRNAs provided herein, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound in which an RNA mimetic that has been shown to have excellent hybridization properties is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative US patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506. The native phosphodiester backbone can be represented as O—P(O)(OH)—OCH2-.

Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m),CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]2, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₃)₂. Further exemplary modifications include: 5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides, 5′-Me-2′-deoxynucleotides, (both R and S isomers in these three families); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative US patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as deoxythimidine (dT), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. Patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

An iRNA agent of the disclosure can also be modified to include one or more bicyclic sugar moieties. A “bicyclic sugar” is a furanosyl ring modified by a ring formed by the bridging of two carbons, whether adjacent or non-adjacent. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a ring formed by bridging two carbons, whether adjacent or non-adjacent, of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring, optionally, via the 2′-acyclic oxygen atom. Thus, in some embodiments an agent of the invention may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4′-CH₂—O-2′ bridge. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the invention include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the invention include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge.

A locked nucleoside can be represented by the structure (omitting stereochemistry),

wherein B is a nucleobase or modified nucleobase and L is the linking group that joins the 2′-carbon to the 4′-carbon of the ribose ring.

Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH₂)—O-2′ (LNA); 4′-(CH₂)—S-2′; 4′-(CH₂)₂—O-2′ (ENA); 4′-CH(CH₃)—O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH₂OCH₃)—O-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH₃)(CH₃)—O-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH₂—N(OCH₃)-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH₂—O—N(CH₃)-2′ (see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH₂—N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a nitrogen protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH₂—C(H)(CH₃)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH₂—C(H₂)-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

Additional representative U.S. Patents and U.S. Patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

The RNA of an iRNA can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH₃)—O-2′ bridge (i.e., L in the preceding structure). In one embodiment, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”

An iRNA of the invention may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the C3 and —C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US2013/0190383; and WO2013/036868, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, an iRNA of the invention comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated by reference).

Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US2013/0096289; US2013/0011922; and US2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in WO2011/005861.

Other modifications of the nucleotides of an iRNA of the invention include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of an iRNA. Suitable phosphate mimics are disclosed in, for example US2012/0157511, the entire contents of which are incorporated herein by reference.

A. Modified iRNAs Comprising Motifs of the Invention

In certain aspects of the invention, the double stranded RNA agents of the invention include agents with chemical modifications as disclosed, for example, in WO2013/075035, the entire contents of each of which are incorporated herein by reference. As shown herein and in WO2013/075035, one or more motifs of three identical modifications on three consecutive nucleotides may be introduced into a sense strand or antisense strand of a dsRNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the dsRNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense or antisense strand. The dsRNAi agent may be optionally conjugated with a GalNAc derivative ligand, for instance on the sense strand.

More specifically, when the sense strand and antisense strand of the double stranded RNA agent are completely modified to have one or more motifs of three identical modifications on three consecutive nucleotides at or near the cleavage site of at least one strand of a dsRNAi agent, the gene silencing activity of the dsRNAi agent was observed.

Accordingly, the invention provides double stranded RNA agents capable of inhibiting the expression of a target gene (i.e., XDH gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be, for example, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”), also referred to herein as “dsRNAi agent.” The duplex region of a dsRNAi agent may be, for example, the duplex region can be 27-30 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In certain embodiments, the dsRNAi agent may contain one or more overhang regions or capping groups at the 3′-end, 5′-end, or both ends of one or both strands. The overhang can be, independently, 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. In certain embodiments, the overhang regions can include extended overhang regions as provided above. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In certain embodiments, the nucleotides in the overhang region of the dsRNAi agent can each independently be a modified or unmodified nucleotide including, but no limited to 2′-sugar modified, such as, 2′-F, 2′-O-methyl, thymidine (T), 2′-O-methoxyethyl-5-methyluridine (Teo), 2′-O-methoxyethyladenosine (Aeo), 2′-O-methoxyethyl-5-methylcytidine (m5Ceo), and any combinations thereof.

For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand, or both strands of the dsRNAi agent may be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In some embodiments, the overhang is present at the 3′-end of the sense strand, antisense strand, or both strands. In some embodiments, this 3′-overhang is present in the antisense strand. In some embodiments, this 3′-overhang is present in the sense strand.

The dsRNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, without affecting its overall stability. For example, the single-stranded overhang may be located at the 3′-end of the sense strand or, alternatively, at the 3′-end of the antisense strand. The RNAi may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the dsRNAi agent has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not wishing to be bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process.

In certain embodiments, the dsRNAi agent is a double blunt-ended of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 7, 8, and 9 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5′end.

In other embodiments, the dsRNAi agent is a double blunt-ended of 20 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 8, 9, and 10 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5′ end.

In yet other embodiments, the dsRNAi agent is a double blunt-ended of 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5′end.

In certain embodiments, the dsRNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, and 11 from the 5′end; the antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, and 13 from the 5′end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nucleotide overhang. In some embodiments, the 2 nucleotide overhang is at the 3′-end of the antisense strand.

When the 2 nucleotide overhang is at the 3′-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand. In certain embodiments, every nucleotide in the sense strand and the antisense strand of the dsRNAi agent, including the nucleotides that are part of the motifs are modified nucleotides. In certain embodiments each residue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif. Optionally, the dsRNAi agent further comprises a ligand (such as, GalNAc).

In certain embodiments, the dsRNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3 ‘ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3’ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In certain embodiments, the dsRNAi agent comprises sense and antisense strands, wherein the dsRNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at position 11, 12, and 13 from the 5′ end; wherein the 3′ end of the first strand and the 5′ end of the second strand form a blunt end and the second strand is 1˜4 nucleotides longer at its 3′ end than the first strand, wherein the duplex region which is at least 25 nucleotides in length, and the second strand is sufficiently complementary to a target mRNA along at least 19 nucleotide of the second strand length to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein Dicer cleavage of the dsRNAi agent results in an siRNA comprising the 3′-end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the dsRNAi agent further comprises a ligand.

In certain embodiments, the sense strand of the dsRNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.

In certain embodiments, the antisense strand of the dsRNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.

For a dsRNAi agent having a duplex region of 19-23 nucleotides in length, the cleavage site of the antisense strand is typically around the 10, 11, and 12 positions from the 5′-end. Thus the motifs of three identical modifications may occur at the 9, 10, and 11 positions; the 10, 11, and 12 positions; the 11, 12, and 13 positions; the 12, 13, and 14 positions; or the 13, 14, and 15 positions of the antisense strand, the count starting from the first nucleotide from the 5′-end of the antisense strand, or, the count starting from the first paired nucleotide within the duplex region from the 5′-end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the dsRNAi agent from the 5′-end.

The sense strand of the dsRNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In some embodiments, the sense strand of the dsRNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand and the other motifs may be a wing modification. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other then the chemistries of the motifs are distinct from each other, and when the motifs are separated by one or more nucleotide than the chemistries can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, each wing modification may occur at one end relative to the first motif which is at or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the dsRNAi agent may contain more than one motifs of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that may be present on the sense strand.

In some embodiments, the wing modification on the sense strand or antisense strand of the dsRNAi agent typically does not include the first one or two terminal nucleotides at the 3′-end, 5′-end, or both ends of the strand.

In other embodiments, the wing modification on the sense strand or antisense strand of the dsRNAi agent typically does not include the first one or two paired nucleotides within the duplex region at the 3′-end, 5′-end, or both ends of the strand.

When the sense strand and the antisense strand of the dsRNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two, or three nucleotides.

When the sense strand and the antisense strand of the dsRNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can be so aligned that two modifications each from one strand fall on one end of the duplex region, having an overlap of one, two, or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides; two modifications one strand fall on each side of the lead motif, having an overlap of one, two or three nucleotides in the duplex region.

In some embodiments, every nucleotide in the sense strand and antisense strand of the dsRNAi agent, including the nucleotides that are part of the motifs, may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′-hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′- or 5′ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of a RNA. For example, a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′-end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′- or 3′-overhang, or in both. For example, it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′- or 5′-overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

In some embodiments, each residue of the sense strand and antisense strand is independently modified with LNA, CRN, cET, UNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, 2′-hydroxyl, or 2′-fluoro. The strands can contain more than one modification. In one embodiment, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro.

At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-O-methyl or 2′-fluoro modifications, or others.

In certain embodiments, the N_(a) or N_(b) comprise modifications of an alternating pattern. The term “alternating motif” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc.

The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In some embodiments, the dsRNAi agent of the invention comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′ to 3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 5′ to 3′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′ to 3′ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 5′ to 3′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

In some embodiments, the dsRNAi agent comprises the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the sense strand initially has a shift relative to the pattern of the alternating motif of 2′-O-methyl modification and 2′-F modification on the antisense strand initially, i.e., the 2′-O-methyl modified nucleotide on the sense strand base pairs with a 2′-F modified nucleotide on the antisense strand and vice versa. The 1 position of the sense strand may start with the 2′-F modification, and the 1 position of the antisense strand may start with the 2′-O-methyl modification.

The introduction of one or more motifs of three identical modifications on three consecutive nucleotides to the sense strand or antisense strand interrupts the initial modification pattern present in the sense strand or antisense strand. This interruption of the modification pattern of the sense or antisense strand by introducing one or more motifs of three identical modifications on three consecutive nucleotides to the sense or antisense strand may enhance the gene silencing activity against the target gene.

In some embodiments, when the motif of three identical modifications on three consecutive nucleotides is introduced to any of the strands, the modification of the nucleotide next to the motif is a different modification than the modification of the motif. For example, the portion of the sequence containing the motif is “ . . . N_(a)YYYN_(b) . . . ,” where “Y” represents the modification of the motif of three identical modifications on three consecutive nucleotide, and “N_(a)” and “N_(b)” represent a modification to the nucleotide next to the motif “YYY” that is different than the modification of Y, and where N_(a) and N_(b) can be the same or different modifications. Alternatively, N_(a) or N_(b) may be present or absent when there is a wing modification present.

The iRNA may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand, antisense strand, or both strands in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand may contain both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand. In one embodiment, a double-stranded RNAi agent comprises 6-8 phosphorothioate internucleotide linkages. In some embodiments, the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-end and two phosphorothioate internucleotide linkages at the 3′-end, and the sense strand comprises at least two phosphorothioate internucleotide linkages at either the 5′-end or the 3′-end.

In some embodiments, the dsRNAi agent comprises a phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region may contain two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within the duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. These terminal three nucleotides may be at the 3′-end of the antisense strand, the 3′-end of the sense strand, the 5′-end of the antisense strand, or the 5′ end of the antisense strand.

In some embodiments, the 2-nucleotide overhang is at the 3′-end of the antisense strand, and there are two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. Optionally, the dsRNAi agent may additionally have two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand.

In one embodiment, the dsRNAi agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch may occur in the overhang region or the duplex region. The base pair may be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In certain embodiments, the dsRNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand independently selected from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In certain embodiments, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2, or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

In other embodiments, the nucleotide at the 3′-end of the sense strand is deoxythimidine (dT) or the nucleotide at the 3′-end of the antisense strand is deoxythimidine (dT). For example, there is a short sequence of deoxythimidine nucleotides, for example, two dT nucleotides on the 3′-end of the sense, antisense strand, or both strands.

In certain embodiments, the sense strand sequence may be represented by formula (I): 5′n _(p)-N_(a)-(XXX)_(i)—N_(b)-YYY-N_(b)-(ZZZ)_(j)—N_(a)-n _(q)3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein N_(b) and Y do not have the same modification; and

XXX, YYY, and ZZZ each independently represent one motif of three identical modifications on three consecutive nucleotides. In some embodiments, YYY is all 2′-F modified nucleotides.

In some embodiments, the N_(a) or N_(b) comprises modifications of alternating pattern.

In some embodiments, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the dsRNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8; 7, 8, 9; 8, 9, 10; 9, 10, 11; 10, 11,12; or 11, 12, 13) of the sense strand, the count starting from the first nucleotide, from the 5′-end; or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas: 5′n _(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′  (Ib); 5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n _(q)3′  (Ic); or 5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′  (Id).

When the sense strand is represented by formula (Ib), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Id), each N_(b) independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. In some embodiments, N_(b) is 0, 1, 2, 3, 4, 5, or 6. Each N_(a) can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula: 5′n _(p)-N_(a)-YYY-N_(a)-n _(q)3′  (Ia).

When the sense strand is represented by formula (Ia), each N_(a) independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (II): 5′n _(q′)-N_(a)′-(Z′Z′Z′)_(k)—N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)_(l)-N′_(a)-n _(p)′3′  (II)

wherein:

k and l are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification; and

X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In some embodiments, the N_(a)′ or N_(b)′ comprises modifications of alternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the dsRNAi agent has a duplex region of 17-23 nucleotides in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the first nucleotide, from the 5′-end; or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end. In some embodiments, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In certain embodiments, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In certain embodiments, k is 1 and 1 is 0, or k is 0 and l is 1, or both k and l are 1.

The antisense strand can therefore be represented by the following formulas: 5′n _(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n _(p′),3′  (IIb); 5′n _(q)′-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n _(p)′3′  (IIc); or 5′n _(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N_(a)′-n _(p′)3′  (IId).

When the antisense strand is represented by formula (IIb), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. In some embodiments, N_(b) is 0, 1, 2, 3, 4, 5, or 6.

In other embodiments, k is 0 and 1 is 0 and the antisense strand may be represented by the formula: 5′n _(p′)-N_(a′)-Y′Y′Y′-N_(a′)-n _(q′)3′  (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, CRN, UNA, cEt, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′, and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.

In some embodiments, the sense strand of the dsRNAi agent may contain YYY motif occurring at 9, 10, and 11 positions of the strand when the duplex region is 21 nt, the count starting from the first nucleotide from the 5′-end, or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.

In some embodiments the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the first nucleotide from the 5′-end, or optionally, the count starting at the first paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with an antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.

Accordingly, the dsRNAi agents for use in the methods of the invention may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the iRNA duplex represented by formula (III): sense: 5′n _(p)-N_(a)-(XXX)_(i)-N_(b)-YYY-N_(b)-(ZZZ)_(j)—N_(a)-n _(q)3′ antisense: 3′n _(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)—N_(a)′-n _(q)′5′  (III)

wherein:

j, k, and l are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotide sequence comprising 0-25 modified nucleotides, each sequence comprising at least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotide sequence comprising 0-10 modified nucleotides;

wherein each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may not be present, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently represent one motif of three identical modifications on three consecutive nucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forming an iRNA duplex include the formulas below: 5′n _(p)-N_(a)-YYY-N_(a)-n _(q)3′ 3′n _(p)′-N_(a)-Y′Y′Y′-N_(a) ′n _(q)′5′  (IIIa) 5′n _(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′ 3′n _(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a) ′n _(q)′5′  (IIIb) 5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n _(q)3′ 3′n _(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n _(q)′5′   (IIIc) 5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′ 3′n _(p)′-N_(a)′-X¹X¹X¹-N_(b)′-Y¹Y¹Y¹-N_(b)′-Z¹Z¹Z¹-N_(a)-n _(q)′5′   (IIId)

When the dsRNAi agent is represented by formula (IIIa), each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented by formula (IIIb), each N_(b) independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5, or 1-4 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented as formula (IIIc), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the dsRNAi agent is represented as formula (IIId), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2, or 0 modified nucleotides. Each N_(a), N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b), and N_(b)′; independently comprises modifications of alternating pattern.

Each of X, Y, and Z in formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) may be the same or different from each other.

When the dsRNAi agent is represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), at least one of the Y nucleotides may form a base pair with one of the Y′ nucleotides. Alternatively, at least two of the Y nucleotides form base pairs with the corresponding Y′ nucleotides; or all three of the Y nucleotides all form base pairs with the corresponding Y′ nucleotides.

When the dsRNAi agent is represented by formula (IIIb) or (IIId), at least one of the Z nucleotides may form a base pair with one of the Z′ nucleotides. Alternatively, at least two of the Z nucleotides form base pairs with the corresponding Z′ nucleotides; or all three of the Z nucleotides all form base pairs with the corresponding Z′ nucleotides.

When the dsRNAi agent is represented as formula (IIIc) or (IIId), at least one of the X nucleotides may form a base pair with one of the X′ nucleotides. Alternatively, at least two of the X nucleotides form base pairs with the corresponding X′ nucleotides; or all three of the X nucleotides all form base pairs with the corresponding X′ nucleotides.

In certain embodiments, the modification on the Y nucleotide is different than the modification on the Y′ nucleotide, the modification on the Z nucleotide is different than the modification on the Z′ nucleotide, or the modification on the X nucleotide is different than the modification on the X′ nucleotide.

In certain embodiments, when the dsRNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. In other embodiments, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications and n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In yet other embodiments, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker (described below). In other embodiments, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In some embodiments, when the dsRNAi agent is represented by formula (IIIa), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more GalNAc derivatives attached through a bivalent or trivalent branched linker.

In some embodiments, the dsRNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In some embodiments, the dsRNAi agent is a multimer containing three, four, five, six, or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, two dsRNAi agents represented by at least one of formulas (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, and one or both of the 3′ ends, and are optionally conjugated to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.

In certain embodiments, an RNAi agent of the invention may contain a low number of nucleotides containing a 2′-fluoro modification, e.g., 10 or fewer nucleotides with 2′-fluoro modification. For example, the RNAi agent may contain 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 0 nucleotides with a 2′-fluoro modification. In a specific embodiment, the RNAi agent of the invention contains 10 nucleotides with a 2′-fluoro modification, e.g., 4 nucleotides with a 2′-fluoro modification in the sense strand and 6 nucleotides with a 2′-fluoro modification in the antisense strand. In another specific embodiment, the RNAi agent of the invention contains 6 nucleotides with a 2′-fluoro modification, e.g., 4 nucleotides with a 2′-fluoro modification in the sense strand and 2 nucleotides with a 2′-fluoro modification in the antisense strand.

In other embodiments, an RNAi agent of the invention may contain an ultra low number of nucleotides containing a 2′-fluoro modification, e.g., 2 or fewer nucleotides containing a 2′-fluoro modification. For example, the RNAi agent may contain 2, 1 of 0 nucleotides with a 2′-fluoro modification. In a specific embodiment, the RNAi agent may contain 2 nucleotides with a 2′-fluoro modification, e.g., 0 nucleotides with a 2-fluoro modification in the sense strand and 2 nucleotides with a 2′-fluoro modification in the antisense strand.

Various publications describe multimeric iRNAs that can be used in the methods of the invention. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520 the entire contents of each of which are hereby incorporated herein by reference.

In certain embodiments, the compositions and methods of the disclosure include a vinyl phosphonate (VP) modification of an RNAi agent as described herein. In exemplary embodiments, a 5′-vinyl phosphonate modified nucleotide of the disclosure has the structure:

wherein X is O or S;

R is hydrogen, hydroxy, fluoro, or C₁₋₂₀alkoxy (e.g., methoxy or n-hexadecyloxy);

R^(5′) is ═C(H)-P(O)(OH)₂ and the double bond between the C5′ carbon and R^(5′) is in the E or Z orientation (e.g., E orientation); and

B is a nucleobase or a modified nucleobase, optionally where B is adenine, guanine, cytosine, thymine, or uracil.

A vinyl phosphonate of the instant disclosure may be attached to either the antisense or the sense strand of a dsRNA of the disclosure. In certain embodiments, a vinyl phosphonate of the instant disclosure is attached to the antisense strand of a dsRNA, optionally at the 5′ end of the antisense strand of the dsRNA.

Vinyl phosphonate modifications are also contemplated for the compositions and methods of the instant disclosure. An exemplary vinyl phosphonate structure includes the preceding structure, where R5′ is ═C(H)—OP(O)(OH)2 and the double bond between the C5′ carbon and R5′ is in the E or Z orientation (e.g., E orientation).

As described in more detail below, the iRNA that contains conjugations of one or more carbohydrate moieties to an iRNA can optimize one or more properties of the iRNA. In many cases, the carbohydrate moiety will be attached to a modified subunit of the iRNA. For example, the ribose sugar of one or more ribonucleotide subunits of an iRNA can be replaced with another moiety, e.g., a non-carbohydrate (such as, cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” such as, two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

The iRNA may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group. In some embodiments, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin. In some embodiments, the acyclic group is a serinol backbone or diethanolamine backbone.

i. Thermally Destabilizing Modifications

In certain embodiments, a dsRNA molecule can be optimized for RNA interference by incorporating thermally destabilizing modifications in the seed region of the antisense strand. As used herein “seed region” means at positions 2-9 of the 5′-end of the referenced strand. For example, thermally destabilizing modifications can be incorporated in the seed region of the antisense strand to reduce or inhibit off-target gene silencing.

The term “thermally destabilizing modification(s)” includes modification(s) that would result with a dsRNA with a lower overall melting temperature (Tm) than the Tm of the dsRNA without having such modification(s). For example, the thermally destabilizing modification(s) can decrease the Tm of the dsRNA by 1-4° C., such as one, two, three or four degrees Celcius. And, the term “thermally destabilizing nucleotide” refers to a nucleotide containing one or more thermally destabilizing modifications.

It has been discovered that dsRNAs with an antisense strand comprising at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5′ end, of the antisense strand have reduced off-target gene silencing activity. Accordingly, in some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region of the antisense strand. In some embodiments, one or more thermally destabilizing modification(s) of the duplex is/are located in positions 2-9, such as, positions 4-8, from the 5′-end of the antisense strand. In some further embodiments, the thermally destabilizing modification(s) of the duplex is/are located at position 6, 7 or 8 from the 5′-end of the antisense strand. In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5′-end of the antisense strand. In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5 or 9 from the 5′-end of the antisense strand.

An iRNA agent comprises a sense strand and an antisense strand, each strand having 14 to 40 nucleotides. The RNAi agent may be represented by formula (L):

In formula (L), B1, B2, B3, B1′, B2′, B3′, and B4′ each are independently a nucleotide containing a modification selected from the group consisting of 2′-O-alkyl, 2′-substituted alkoxy, 2′-substituted alkyl, 2′-halo, ENA, and BNA/LNA. In one embodiment, B1, B2, B3, B1′, B2′, B3′, and B4′ each contain 2′-OMe modifications. In one embodiment, B1, B2, B3, B1′, B2′, B3′, and B4′ each contain 2′-OMe or 2′-F modifications. In one embodiment, at least one of B1, B2, B3, B1′, B2′, B3′, and B4′ contain 2′-O-N-methylacetamido (2′-O-NMA, 2′O-CH₂C(O)N(Me)H) modification.

C1 is a thermally destabilizing nucleotide placed at a site opposite to the seed region of the antisense strand (i.e., at positions 2-8 of the 5′-end of the antisense strand). For example, C1 is at a position of the sense strand that pairs with a nucleotide at positions 2-8 of the 5′-end of the antisense strand. In one example, C1 is at position 15 from the 5′-end of the sense strand. C1 nucleotide bears the thermally destabilizing modification which can include abasic modification; mismatch with the opposing nucleotide in the duplex; and sugar modification such as 2′-deoxy modification or acyclic nucleotide e.g., unlocked nucleic acids (UNA) or glycerol nucleic acid (GNA). In one embodiment, C1 has thermally destabilizing modification selected from the group consisting of: i) mismatch with the opposing nucleotide in the antisense strand; ii) abasic modification selected from the group consisting of:

and iii) sugar modification selected from the group consisting of:

wherein B is a modified or unmodified nucleobase, R¹ and R² independently are H, halogen, OR₃, or alkyl; and R3 is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar. In one embodiment, the thermally destabilizing modification in C1 is a mismatch selected from the group consisting of G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, and U:T; and optionally, at least one nucleobase in the mismatch pair is a 2′-deoxy nucleobase. In one example, the thermally destabilizing modification in C1 is GNA or

T1, T1′, T2′, and T3′ each independently represent a nucleotide comprising a modification providing the nucleotide a steric bulk that is less or equal to the steric bulk of a 2′-OMe modification. A steric bulk refers to the sum of steric effects of a modification. Methods for determining steric effects of a modification of a nucleotide are known to one skilled in the art. The modification can be at the 2′ position of a ribose sugar of the nucleotide, or a modification to a non-ribose nucleotide, acyclic nucleotide, or the backbone of the nucleotide that is similar or equivalent to the 2′ position of the ribose sugar, and provides the nucleotide a steric bulk that is less than or equal to the steric bulk of a 2′-OMe modification. For example, T1, T1′, T2′, and T3′ are each independently selected from DNA, RNA, LNA, 2′-F, and 2′-F-5′-methyl. In one embodiment, T1 is DNA. In one embodiment, T1′ is DNA, RNA or LNA. In one embodiment, T2′ is DNA or RNA. In one embodiment, T3′ is DNA or RNA. n¹, n³, and q¹ are independently 4 to 15 nucleotides in length. n⁵, q³, and q⁷ are independently 1-6 nucleotide(s) in length. n⁴, q², and q⁶ are independently 1-3 nucleotide(s) in length; alternatively, n⁴ is 0. q⁵ is independently 0-10 nucleotide(s) in length. n² and q⁴ are independently 0-3 nucleotide(s) in length.

Alternatively, n⁴ is 0-3 nucleotide(s) in length.

In one embodiment, n⁴ can be 0. In one example, n⁴ is 0, and q² and q⁶ are 1. In another example, n⁴ is 0, and q² and q⁶ are 1, with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, n⁴, q², and q⁶ are each 1.

In one embodiment, n², n⁴, q², q⁴, and q⁶ are each 1.

In one embodiment, C1 is at position 14-17 of the 5′-end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n⁴ is 1. In one embodiment, C1 is at position 15 of the 5′-end of the sense strand

In one embodiment, T3′ starts at position 2 from the 5′ end of the antisense strand. In one example, T3′ is at position 2 from the 5′ end of the antisense strand and q⁶ is equal to 1.

In one embodiment, T1′ starts at position 14 from the 5′ end of the antisense strand. In one example, T1′ is at position 14 from the 5′ end of the antisense strand and q² is equal to 1.

In an exemplary embodiment, T3′ starts from position 2 from the 5′ end of the antisense strand and T1′ starts from position 14 from the 5′ end of the antisense strand. In one example, T3′ starts from position 2 from the 5′ end of the antisense strand and q⁶ is equal to 1 and T1′ starts from position 14 from the 5′ end of the antisense strand and q² is equal to 1.

In one embodiment, T1′ and T3′ are separated by 11 nucleotides in length (i.e. not counting the T1′ and T3′ nucleotides).

In one embodiment, T1′ is at position 14 from the 5′ end of the antisense strand. In one example, T1′ is at position 14 from the 5′ end of the antisense strand and q² is equal to 1, and the modification at the 2′ position or positions in a non-ribose, acyclic or backbone that provide less steric bulk than a 2′-OMe ribose.

In one embodiment, T3′ is at position 2 from the 5′ end of the antisense strand. In one example, T3′ is at position 2 from the 5′ end of the antisense strand and q⁶ is equal to 1, and the modification at the 2′ position or positions in a non-ribose, acyclic or backbone that provide less than or equal to steric bulk than a 2′-OMe ribose.

In one embodiment, T1 is at the cleavage site of the sense strand. In one example, T1 is at position 11 from the 5′ end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n² is 1. In an exemplary embodiment, T1 is at the cleavage site of the sense strand at position 11 from the 5′ end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n² is 1, In one embodiment, T2′ starts at position 6 from the 5′ end of the antisense strand. In one example, T2′ is at positions 6-10 from the 5′ end of the antisense strand, and q⁴ is 1.

In an exemplary embodiment, T1 is at the cleavage site of the sense strand, for instance, at position 11 from the 5′ end of the sense strand, when the sense strand is 19-22 nucleotides in length, and n² is 1; T1′ is at position 14 from the 5′ end of the antisense strand, and q² is equal to 1, and the modification to T1′ is at the 2′ position of a ribose sugar or at positions in a non-ribose, acyclic or backbone that provide less steric bulk than a 2′-OMe ribose; T2′ is at positions 6-10 from the 5′ end of the antisense strand, and q⁴ is 1; and T3′ is at position 2 from the 5′ end of the antisense strand, and q⁶ is equal to 1, and the modification to T3′ is at the 2′ position or at positions in a non-ribose, acyclic or backbone that provide less than or equal to steric bulk than a 2′-OMe ribose.

In one embodiment, T2′ starts at position 8 from the 5′ end of the antisense strand. In one example, T2′ starts at position 8 from the 5′ end of the antisense strand, and q⁴ is 2.

In one embodiment, T2′ starts at position 9 from the 5′ end of the antisense strand. In one example, T2′ is at position 9 from the 5′ end of the antisense strand, and q⁴ is 1. In one embodiment, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 6, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 7, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 6, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 7, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 6, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 5, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; optionally with at least 2 additional TT at the 3′-end of the antisense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 5, T2′ is 2′-F, q⁴ is 1, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; optionally with at least 2 additional TT at the 3′-end of the antisense strand; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within positions 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand).

The RNAi agent can comprise a phosphorus-containing group at the 5′-end of the sense strand or antisense strand. The 5′-end phosphorus-containing group can be 5′-end phosphate (5′-P), 5′-end phosphorothioate (5′-PS), 5′-end phosphorodithioate (5′-PS₂), 5′-end vinylphosphonate (5′-VP), 5′-end methylphosphonate (MePhos), or 5′-deoxy-5′-C-malonyl

When the 5′-end phosphorus-containing group is 5′-end vinylphosphonate (5′-VP), the 5′-VP can be either 5′-E-VP isomer (i.e., trans-vinylphosphonate,

5′-Z-VP isomer (i.e., cis-vinylphosphonate,

or mixtures thereof. In one embodiment, the RNAi agent comprises a phosphorus-containing group at the 5′-end of the sense strand. In one embodiment, the RNAi agent comprises a phosphorus-containing group at the 5′-end of the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-P. In one embodiment, the RNAi agent comprises a 5′-P in the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-PS. In one embodiment, the RNAi agent comprises a 5′-PS in the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-VP. In one embodiment, the RNAi agent comprises a 5′-VP in the antisense strand. In one embodiment, the RNAi agent comprises a 5′-E-VP in the antisense strand. In one embodiment, the RNAi agent comprises a 5′-Z-VP in the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-PS₂. In one embodiment, the RNAi agent comprises a 5′-PS₂ in the antisense strand.

In one embodiment, the RNAi agent comprises a 5′-PS₂. In one embodiment, the RNAi agent comprises a 5′-deoxy-5′-C-malonyl in the antisense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The dsRNA agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1. The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The dsRNAi RNA agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1. The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP. The 5′-VP may be 5′-E-VP, 5′-Z-VP, or combination thereof.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P and a targeting ligand. In one embodiment, the 5′-P is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS and a targeting ligand. In one embodiment, the 5′-PS is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, or combination thereof), and a targeting ligand.

In one embodiment, the 5′-VP is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂ and a targeting ligand. In one embodiment, the 5′-PS₂ is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and a targeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-P and a targeting ligand. In one embodiment, the 5′-P is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-PS and a targeting ligand. In one embodiment, the 5′-PS is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, or combination thereof) and a targeting ligand. In one embodiment, the 5′-VP is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-PS₂ and a targeting ligand. In one embodiment, the 5′-PS₂ is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-OMe, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and a targeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P and a targeting ligand. In one embodiment, the 5′-P is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS and a targeting ligand. In one embodiment, the 5′-PS is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, or combination thereof) and a targeting ligand. In one embodiment, the 5′-VP is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂ and a targeting ligand. In one embodiment, the 5′-PS₂ is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, BF is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′- F, q³ is 4, T2′ is 2′-F, q⁴ is 2, B3′ is 2′-OMe or 2′-F, q⁵ is 5, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and a targeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-P and a targeting ligand. In one embodiment, the 5′-P is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS and a targeting ligand. In one embodiment, the 5′-PS is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-VP (e.g., a 5′-E-VP, 5′-Z-VP, or combination thereof) and a targeting ligand. In one embodiment, the 5′-VP is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-PS₂ and a targeting ligand. In one embodiment, the 5′-PS₂ is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In one embodiment, B1 is 2′-OMe or 2′-F, n¹ is 8, T1 is 2′F, n² is 3, B2 is 2′-OMe, n³ is 7, n⁴ is 0, B3 is 2′-OMe, n⁵ is 3, B1′ is 2′-OMe or 2′-F, q¹ is 9, T1′ is 2′-F, q² is 1, B2′ is 2′-OMe or 2′-F, q³ is 4, q⁴ is 0, B3′ is 2′-OMe or 2′-F, q⁵ is 7, T3′ is 2′-F, q⁶ is 1, B4′ is 2′-F, and q⁷ is 1; with two phosphorothioate internucleotide linkage modifications within position 1-5 of the sense strand (counting from the 5′-end of the sense strand), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end of the antisense strand). The RNAi agent also comprises a 5′-deoxy-5′-C-malonyl and a targeting ligand. In one embodiment, the 5′-deoxy-5′-C-malonyl is at the 5′-end of the antisense strand, and the targeting ligand is at the 3′-end of the sense strand.

In a particular embodiment, an RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker; and         -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11,             13, 17, 19, and 21, and 2′-OMe modifications at positions 2,             4, 6, 8, 12, 14 to 16, 18, and 20 (counting from the 5′             end); and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3, 5, 9, 11 to 13,             15, 17, 19, 21, and 23, and 2′F modifications at positions             2, 4, 6 to 8, 10, 14, 16, 18, 20, and 22 (counting from the             5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 21 and 22,         -   and between nucleotide positions 22 and 23 (counting from             the 5′ end);     -   wherein the dsRNA agents have a two nucleotide overhang at the         3′-end of the antisense strand, and a blunt end at the 5′-end of         the antisense strand.

In another particular embodiment, an RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11,             13, 15, 17, 19, and 21, and 2′-OMe modifications at             positions 2, 4, 6, 8, 12, 14, 16, 18, and 20 (counting from             the 5′ end); and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end); and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3, 5, 7, 9, 11 to             13, 15, 17, 19, and 21 to 23, and 2′F modifications at             positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from             the 5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-OMe modifications at positions 1 to 6, 8, 10, and             12 to 21, 2′-F modifications at positions 7, and 9, and a             deoxy-nucleotide (e.g. dT) at position 11 (counting from the             5′ end); and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end); and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3, 7, 9, 11, 13,             15, 17, and 19 to 23, and 2′-F modifications at positions 2,             4 to 6, 8, 10, 12, 14, 16, and 18 (counting from the 5′             end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-OMe modifications at positions 1 to 6, 8, 10, 12,             14, and 16 to 21, and 2′-F modifications at positions 7, 9,             11, 13, and 15; and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end); and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 5, 7, 9, 11, 13,             15, 17, 19, and 21 to 23, and 2′-F modifications at             positions 2 to 4, 6, 8, 10, 12, 14, 16, 18, and 20 (counting             from the 5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-OMe modifications at positions 1 to 9, and 12 to             21, and 2′-F modifications at positions 10, and 11; and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end); and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3, 5, 7, 9, 11 to             13, 15, 17, 19, and 21 to 23, and 2′-F modifications at             positions 2, 4, 6, 8, 10, 14, 16, 18, and 20 (counting from             the 5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-F modifications at positions 1, 3, 5, 7, 9 to 11,             and 13, and 2′-OMe modifications at positions 2, 4, 6, 8,             12, and 14 to 21; and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end); and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3, 5 to 7, 9, 11             to 13, 15, 17 to 19, and 21 to 23, and 2′-F modifications at             positions 2, 4, 8, 10, 14, 16, and 20 (counting from the 5′             end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-OMe modifications at positions 1, 2, 4, 6, 8, 12,             14, 15, 17, and 19 to 21, and 2′-F modifications at             positions 3, 5, 7, 9 to 11, 13, 16, and 18; and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end); and     -   (b) an antisense strand having:         -   (i) a length of 25 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 4, 6, 7, 9, 11 to             13, 15, 17, and 19 to 23, 2′-F modifications at positions 2,             3, 5, 8, 10, 14, 16, and 18, and desoxy-nucleotides (e.g.             dT) at positions 24 and 25 (counting from the 5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a four nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-OMe modifications at positions 1 to 6, 8, and 12 to             21, and 2′-F modifications at positions 7, and 9 to 11; and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end); and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 8, 10             to 13, 15, and 17 to 23, and 2′-F modifications at positions             2, 6, 9, 14, and 16 (counting from the 5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-OMe modifications at positions 1 to 6, 8, and 12 to             21, and 2′-F modifications at positions 7, and 9 to 11; and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end); and     -   (b) an antisense strand having:         -   (i) a length of 23 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 10 to             13, 15, and 17 to 23, and 2′-F modifications at positions 2,             6, 8, 9, 14, and 16 (counting from the 5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 21 and 22, and between             nucleotide positions 22 and 23 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In another particular embodiment, a RNAi agent of the present invention comprises:

-   -   (a) a sense strand having:         -   (i) a length of 19 nucleotides;         -   (ii) an ASGPR ligand attached to the 3′-end, wherein said             ASGPR ligand comprises three GalNAc derivatives attached             through a trivalent branched linker;         -   (iii) 2′-OMe modifications at positions 1 to 4, 6, and 10 to             19, and 2′-F modifications at positions 5, and 7 to 9; and         -   (iv) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, and between nucleotide             positions 2 and 3 (counting from the 5′ end); and     -   (b) an antisense strand having:         -   (i) a length of 21 nucleotides;         -   (ii) 2′-OMe modifications at positions 1, 3 to 5, 7, 10 to             13, 15, and 17 to 21, and 2′-F modifications at positions 2,             6, 8, 9, 14, and 16 (counting from the 5′ end); and         -   (iii) phosphorothioate internucleotide linkages between             nucleotide positions 1 and 2, between nucleotide positions 2             and 3, between nucleotide positions 19 and 20, and between             nucleotide positions 20 and 21 (counting from the 5′ end);             wherein the RNAi agents have a two nucleotide overhang at             the 3′-end of the antisense strand, and a blunt end at the             5′-end of the antisense strand.

In certain embodiments, the iRNA for use in the methods of the invention is an agent selected from agents listed in any one of Tables 2-3 and 6-7. These agents may further comprise a ligand.

III. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the iRNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the iRNA e.g., into a cell. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556). In other embodiments, the ligand is cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In certain embodiments, a ligand alters the distribution, targeting, or lifetime of an iRNA agent into which it is incorporated. In some embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. In some embodiments, ligands do not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic. In certain embodiments, the ligand is a multivalent galactose, e.g., an N-acetyl-galactosamine.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxol, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins, etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated iRNAs of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems® (Foster City, Calif.). Any other methods for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated iRNAs and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipid Conjugates

In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. In some embodiments, such a lipid or lipid-based molecule binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In certain embodiments, the lipid based ligand binds HSA. In some embodiments, it binds HSA with a sufficient affinity such that the conjugate will be distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In other embodiments, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be distributed to the kidney. Other moieties that target to kidney cells can also be used in place of, or in addition to, the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, such as, a helical cell-permeation agent. In some embodiments, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. In some embodiments, the helical agent is an alpha-helical agent, which has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 17). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 18) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 19) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 20) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand, e.g., PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, an iRNA further comprises a carbohydrate. The carbohydrate conjugated iRNA is advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri-, and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In certain embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide.

In certain embodiments, the monosaccharide is an N-acetylgalactosamine (GalNAc). GalNAc conjugates, which comprise one or more N-acetylgalactosamine (GalNAc) derivatives, are described, for example, in U.S. Pat. No. 8,106,022, the entire content of which is hereby incorporated herein by reference.

In some embodiments, the GalNAc conjugate serves as a ligand that targets the iRNA to particular cells. In some embodiments, the GalNAc conjugate targets the iRNA to liver cells, e.g., by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be attached via a linker, e.g., a bivalent or trivalent branched linker. In some embodiments the GalNAc conjugate is conjugated to the 3′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3′ end of the sense strand) via a linker, e.g., a linker as described herein. In some embodiments the GalNAc conjugate is conjugated to the 5′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5′ end of the sense strand) via a linker, e.g., a linker as described herein.

In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker. In other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a tetravalent linker.

In certain embodiments, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent. In certain embodiments, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.

In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

wherein Y is O or S and n is 3-6 (Formula XXIV);

wherein Y is O or S and n is 3-6 (Formula XXV);

wherein X is O or S (Formula XXVII);

In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as

In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic, wherein X is O or S

In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below:

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

(Formula XXXVI), when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, a suitable ligand is a ligand disclosed in WO 2019/055633, the entire contents of which are incorporated herein by reference. In one embodiment the ligand comprises the structure below:

In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker.

In one embodiment, the double stranded RNAi agents of the invention comprise one or more GalNAc or GalNAc derivative attached to the iRNA agent. The GalNAc may be attached to any nucleotide via a linker on the sense strand or antsisense strand. The GalNac may be attached to the 5′-end of the sense strand, the 3′ end of the sense strand, the 5′-end of the antisense strand, or the 3′-end of the antisense strand. In one embodiment, the GalNAc is attached to the 3′ end of the sense strand, e.g., via a trivalent linker.

In other embodiments, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of linkers, e.g., monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agent of the invention is part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell permeation peptide.

Additional carbohydrate conjugates and linkers suitable for use in the present invention include those described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NRB, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic, or substituted aliphatic. In one embodiment, the linker is about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In one embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or more, or at least 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential, or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a selected pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In some embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

i. Redox cleavable linking groups

In certain embodiments, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

ii. Phosphate-based cleavable linking groups

In other embodiments, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—, wherein Rk at each occurrence can be, independently, C1-C20 alkyl, C1-C20 haloalkyl, C6-C10 aryl, or C7-C12 aralkyl. Exemplary embodiments include are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—, —S—P(O)(H)—S—, and —O—P(S)(H)—S—. In certain embodiments, a phosphate-based linking group is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

iii. Acid cleavable linking groups

In other embodiments, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In some embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). An exemplary embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

iv. Ester-based linking groups

In other embodiments, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include, but are not limited to, esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

v. Peptide-based cleaving groups

In yet other embodiments, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH-). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

In some embodiments, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,

(Formula XLIV), when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention, a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XLV)-(XLVIII):

wherein: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5 q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; p^(2A), p^(2B), p^(3A), p^(3B), p^(4A), p^(4B), p^(5A), p^(5B), p^(5C), T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C) are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O; Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C) are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), CEC or C(O); R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C) are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO, CH═N—O.

or heterocyclyl; L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) and L^(5C) represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XLIX):

wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide, such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. Patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, such as, dsRNAi agents, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

IV. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject susceptible to or diagnosed with a xanthine dehydrogenase-associated disorder) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian R L. (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids 32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al (2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience 129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602). Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., et al (2004) Nature 432:173-178).

In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H, et al (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R, et al (2003) J. Mol. Biol 327:761-766; Verma, U N, et al (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N, et al (2003), supra), “solid nucleic acid lipid particles” (Zimmermann, T S, et al (2006) Nature 441:111-114), cardiolipin (Chien, P Y, et al (2005) Cancer Gene Ther. 12:321-328; Pal, A, et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E, et al (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A, et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., et al (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

A. Vector encoded iRNAs of the Invention

iRNA targeting the xanthine dehydrogenase gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A, et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA (1995) 92:1292).

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are known in the art.

V. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA are useful for preventing or treating a xanthine dehydrogenase-associated disorder. Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by subcutaneous (SC), intramuscular (IM), or intravenous (IV) delivery. The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a xanthine dehydrogenase gene.

In some embodiments, the pharmaceutical compositions of the invention are sterile. In another embodiment, the pharmaceutical compositions of the invention are pyrogen free.

The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a xanthine dehydrogenase gene. In general, a suitable dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. Typically, a suitable dose of an iRNA of the invention will be in the range of about 0.1 mg/kg to about 5.0 mg/kg, or about 0.3 mg/kg to about 3.0 mg/kg. A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as every month, once every 3-6 months, or once a year. In certain embodiments, the iRNA is administered about once per month to about once per six months.

After an initial treatment regimen, the treatments can be administered on a less frequent basis. Duration of treatment can be determined based on the severity of disease.

In other embodiments, a single dose of the pharmaceutical compositions can be long lasting, such that doses are administered at not more than 1, 2, 3, or 4 month intervals. In some embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered about once per month. In other embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered quarterly (i.e., about every three months). In other embodiments of the invention, a single dose of the pharmaceutical compositions of the invention is administered twice per year (i.e., about once every six months).

The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to mutations present in the subject, previous treatments, the general health or age of the subject, and other diseases present. Moreover, treatment of a subject with a prophylactically or therapeutically effective amount, as appropriate, of a composition can include a single treatment or a series of treatments.

The iRNA can be delivered in a manner to target a particular tissue (e.g., hepatocytes).

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids, and self-emulsifying semisolids. Formulations include those that target the liver.

The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers.

A. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution either in the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic, and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives, and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

The application of emulsion formulations via dermatological, oral, and parenteral routes, and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil, and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).

iii. Microparticles

An iRNA of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers and their use in manufacture of pharmaceutical compositions and delivery of pharmaceutical agents are well known in the art.

v. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent, or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Such agent are well known in the art.

vi. Other Components

The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings, or aromatic substances, and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol, or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA and (b) one or more agents which function by a non-iRNA mechanism and which are useful in treating a xanthine dehydrogenase-associated disorder.

Toxicity and prophylactic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose prophylactically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED50, such as, an ED80 or ED90, with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the prophylactically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) or higher levels of inhibition as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents used for the prevention or treatment of a xanthine dehydrogenase-associated disorder. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VI. Methods For Inhibiting Xanthine Dehydrogenase Expression

The present invention also provides methods of inhibiting expression of an XDH gene in a cell. The methods include contacting a cell with an RNAi agent, e.g., double stranded RNA agent, in an amount effective to inhibit expression of XDH in the cell, thereby inhibiting expression of XDH in the cell.

Contacting of a cell with an iRNA, e.g., a double stranded RNA agent, may be done in vitro or in vivo. Contacting a cell in vivo with the iRNA includes contacting a cell or group of cells within a subject, e.g., a human subject, with the iRNA. Combinations of in vitro and in vivo methods of contacting a cell are also possible. Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAc ligand, or any other ligand that directs the RNAi agent to a site of interest.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating”, “suppressing”, and other similar terms, and includes any level of inhibition.

The phrase “inhibiting expression of a xanthine dehydrogenase gene” is intended to refer to inhibition of expression of any xanthine dehydrogenase gene (such as, e.g., a mouse xanthine dehydrogenase gene, a rat xanthine dehydrogenase gene, a monkey xanthine dehydrogenase gene, or a human xanthine dehydrogenase gene) as well as variants or mutants of a xanthine dehydrogenase gene. Thus, the xanthine dehydrogenase gene may be a wild-type xanthine dehydrogenase gene, a mutant xanthine dehydrogenase gene, or a transgenic xanthine dehydrogenase gene in the context of a genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of a xanthine dehydrogenase gene” includes any level of inhibition of a xanthine dehydrogenase gene, e.g., at least partial suppression of the expression of a xanthine dehydrogenase gene, such as, a clinically relevant level of supression. The expression of the xanthine dehydrogenase gene may be assessed based on the level, or the change in the level, of any variable associated with xanthine dehydrogenase gene expression, e.g., xanthine dehydrogenase mRNA level or xanthine dehydrogenase protein level, or, for example, serum uric acid levels. Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject. It is understood that xanthine dehydrogenase is expressed predominantly in the liver, and is present in circulation.

Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with xanthine dehydrogenase expression compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

In some embodiments of the methods of the invention, expression of a xanthine dehydrogenase gene is inhibited by at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay. In some embodiments, expression of a xanthine dehydrogenase gene is inhibited by at least 70%. It is further understood that inhibition of xanthine dehydrogenase expression in certain tissues, e.g., in gall bladder, without a significant inhibition of expression in other tissues, e.g., brain, may be desirable. In some embodiments, expression level is determined using the assay method provided in Example 2 with a 10 nM siRNA concentration in the appropriate species matched cell line.

In certain embodiments, inhibition of expression in vivo is determined by knockdown of the human gene in a rodent expressing the human gene, e.g., an AAV-infected mouse expressing the human target gene (i.e., xanthine dehydrogenase), e.g., when administered as a single dose, e.g., at 3 mg/kg at the nadir of RNA expression. Knockdown of expression of an endogenous gene in a model animal system can also be determined, e.g., after administration of a single dose at, e.g., 3 mg/kg at the nadir of RNA expression. Such systems are useful when the nucleic acid sequence of the human gene and the model animal gene are sufficiently close such that the human iRNA provides effective knockdown of the model animal gene. RNA expression in liver is determined using the PCR methods provided in Example 2.

Inhibition of the expression of a xanthine dehydrogenase gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which a xanthine dehydrogenase gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with an iRNA of the invention, or by administering an iRNA of the invention to a subject in which the cells are or were present) such that the expression of a xanthine dehydrogenase gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s) not treated with an iRNA or not treated with an iRNA targeted to the gene of interest). In some embodiments, the inhibition is assessed by the method provided in Example 2 using, e.g., a 10 nM siRNA concentration in the species matched cell line and expressing the level of mRNA in treated cells as a percentage of the level of mRNA in control cells, using the following formula:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

In other embodiments, inhibition of the expression of a xanthine dehydrogenase gene may be assessed in terms of a reduction of a parameter that is functionally linked to xanthine dehydrogenase gene expression, e.g., xanthine dehydrogenase protein level in blood or serum from a subject. Xanthine dehydrogenase gene silencing may be determined in any cell expressing xanthine dehydrogenase, either endogenous or heterologous from an expression construct, and by any assay known in the art.

Inhibition of the expression of a xanthine dehydrogenase protein may be manifested by a reduction in the level of the xanthine dehydrogenase protein that is expressed by a cell or group of cells or in a subject sample (e.g., the level of protein in a blood sample derived from a subject). As explained above, for the assessment of mRNA suppression, the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells, or the change in the level of protein in a subject sample, e.g., blood or serum derived therefrom.

A control cell, a group of cells, or subject sample that may be used to assess the inhibition of the expression of a xanthine dehydrogenase gene includes a cell, group of cells, or subject sample that has not yet been contacted with an RNAi agent of the invention. For example, the control cell, group of cells, or subject sample may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent or an appropriately matched population control.

The level of xanthine dehydrogenase mRNA that is expressed by a cell or group of cells may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of xanthine dehydrogenase in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the xanthine dehydrogenase gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy™ RNA preparation kits (Qiagen®) or PAXgene™ (PreAnalytix™, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis.

In some embodiments, the level of expression of xanthine dehydrogenase is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific xanthine dehydrogenase. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to xanthine dehydrogenase mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix® gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of xanthine dehydrogenase mRNA.

An alternative method for determining the level of expression of xanthine dehydrogenase in a sample involves the process of nucleic acid amplification or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, the level of expression of XDH is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™ System).

In some embodiments, expression level is determined by the method provided in Example 2 using, e.g., a 10 nM siRNA concentration, in the species matched cell line.

The expression levels of xanthine dehydrogenase mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of xanthine dehydrogenase expression level may also comprise using nucleic acid probes in solution.

In some embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of these methods is described and exemplified in the Examples presented herein. In some embodiments, expression level is determined by the method provided in Example 2 using a 10 nM siRNA concentration in the species matched cell line.

The level of XDH protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like.

In some embodiments, the efficacy of the methods of the invention are assessed by a decrease in XDH mRNA or protein level (e.g., in a liver biopsy).

In some embodiments of the methods of the invention, the iRNA is administered to a subject such that the iRNA is delivered to a specific site within the subject. The inhibition of expression of xanthine dehydrogenase may be assessed using measurements of the level or change in the level of xanthine dehydrogenase mRNA or xanthine dehydrogenase protein in a sample derived from fluid or tissue from the specific site within the subject (e.g., liver or blood).

As used herein, the terms detecting or determining a level of an analyte are understood to mean performing the steps to determine if a material, e.g., protein, RNA, is present. As used herein, methods of detecting or determining include detection or determination of an analyte level that is below the level of detection for the method used.

VII. Prophylactic and Treatment Methods of the Invention

The present invention also provides methods of using an iRNA of the invention or a composition containing an iRNA of the invention to inhibit expression of xanthine dehydrogenase, thereby preventing or treating a xanthine dehydrogenase-associated disorder, e.g., hyperuricemia, gout NAFLD, NASH, metabolic disorder, insulin resistance, cardiovascular disease, hypertension, type 2 diabetes, and conditions linked to oxidative stress e.g., chronic low grade inflammation.

In one embodiment, the xanthine dehydrogenase-associate disease is hyperuricemia.

In another embodiment, the xanthine dehydrogenase-associate disease is gout.

In the methods of the invention the cell may be contacted with the siRNA in vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may be any cell that expresses a xanthine dehydrogenase gene, e.g., a liver cell, a brain cell, a gall bladder cell, a heart cell, or a kidney cell. In some embodiment, the cell is a liver cell. A cell suitable for use in the methods of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell, including human cell in a chimeric non-human animal, or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), or a non-primate cell. In certain embodiments, the cell is a human cell, e.g., a human liver cell. In the methods of the invention, xanthine dehydrogenase expression is inhibited in the cell by at least 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95, or to a level below the level of detection of the assay.

The in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the xanthine dehydrogenase gene of the mammal to which the RNAi agent is to be administered. The composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection. In certain embodiments, the compositions are administered by intramuscular injection.

In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of XDH, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In some embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In some embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods for inhibiting the expression of a xanthine dehydrogenase gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets a xanthine dehydrogenase gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the xanthine dehydrogenase gene, thereby inhibiting expression of the xanthine dehydrogenase gene in the cell. Reduction in gene expression can be assessed by any methods known in the art and by methods, e.g. qRT-PCR, described herein, e.g., in Example 2. Reduction in protein production can be assessed by any methods known it the art, e.g. ELISA. In certain embodiments, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in the xanthine dehydrogenase gene or protein expression. In other embodiments, a blood sample serves as the subject sample for monitoring the reduction in the xanthine dehydrogenase protein expression.

The present invention further provides methods of treatment in a subject in need thereof, e.g., a subject diagnosed with a xanthine dehydrogenase-associated disorder, such as, hyperuricemia, gout NAFLD, NASH, metabolic disorder, insulin resistance, cardiovascular disease, hypertension, type 2 diabetes, and conditions linked to oxidative stress e.g., chronic low grade inflammation.

The present invention further provides methods of prophylaxis in a subject in need thereof. The treatment methods of the invention include administering an iRNA of the invention to a subject, e.g., a subject that would benefit from a reduction of xanthine dehydrogenase expression, in a prophylactically effective amount of an iRNA targeting a xanthine dehydrogenase gene or a pharmaceutical composition comprising an iRNA targeting a xanthine dehydrogenase gene.

In one embodiment, the xanthine dehydrogenase-associate disease is hyperuricemia.

In another embodiment, the xanthine dehydrogenase-associate disease is gout.

An iRNA of the invention may be administered as a “free iRNA.” A free iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.

Alternatively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from an inhibition of xanthine dehydrogenase expression are subjects susceptible to or diagnosed with an XDH-associated disorder, e.g., hyperuricemia or gout.

In an embodiment, the method includes administering a composition featured herein such that expression of the target xanthine dehydrogenase gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 1-6, 1-3, or 3-6 months per dose. In certain embodiments, the composition is administered once every 3-6 months.

In some embodiments, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target xanthine dehydrogenase gene. Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein.

Administration of the iRNA according to the methods of the invention may result prevention or treatment of a xanthine dehydrogenase-associated disorder, e.g., hyperuricemia and gout.

Subjects can be administered a therapeutic amount of iRNA, such as about 0.01 mg/kg to about 200 mg/kg. Subjects can be administered a therapeutic amount of iRNA, such as about 5 mg to about 1000 mg as a fixed dose, regardless of body weight.

In some embodiments, the iRNA is administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired dose of iRNA to a subject. The injections may be repeated over a period of time.

The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimen may include administration of a therapeutic amount of iRNA on a regular basis, such as once per month to once a year. In certain embodiments, the iRNA is administered about once per month to about once every three months, or about once every three months to about once every six months.

The invention further provides methods and uses of an iRNA agent or a pharmaceutical composition thereof for treating a subject that would benefit from reduction or inhibition of XDH gene expression, e.g., a subject having an XDH-associated disease, in combination with other pharmaceuticals or other therapeutic methods, e.g., with known pharmaceuticals or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. Exemplary pharmaceuticals include, for example, allopurinol, oxypurinol, Probenecid, Rasburicase, febuxostat, analgesic or anti-inflammatory agents, e.g., NSAIDS.

Accordingly, in some aspects of the invention, the methods which include either a single iRNA agent of the invention, further include administering to the subject one or more additional therapeutic agents. The iRNA agent and an additional therapeutic agent or treatment may be administered at the same time or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times or by another method known in the art or described herein.

In one embodiment, an iRNA agent is administered in combination with allopurinol. In one embodiment, the iRNA agent is administered to the patient, and then the additional therapeutic agent is administered to the patient (or vice versa). In another embodiment, the iRNA agent and the additional therapeutic agent are administered at the same time.

The iRNA agent and an additional therapeutic agent or treatment may be administered at the same time or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times or by another method known in the art or described herein.

VIII. Kits

In certain aspects, the instant disclosure provides kits that include a suitable container containing a pharmaceutical formulation of a siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, or precursor thereof).

Such kits include one or more dsRNA agent(s) and instructions for use, e.g., instructions for administering a prophylactically or therapeutically effective amount of a dsRNA agent(s). The dsRNA agent may be in a vial or a pre-filled syringe. The kits may optionally further comprise means for administering the dsRNA agent (e.g., an injection device, such as a pre-filled syringe), or means for measuring the inhibition of XDH (e.g., means for measuring the inhibition of XDH mRNA, XDH protein, or XDH activity). Such means for measuring the inhibition of XDH may comprise a means for obtaining a sample from a subject, such as, e.g., a plasma sample. The kits of the invention may optionally further comprise means for determining the therapeutically effective or prophylactically effective amount.

In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container, e.g., a vial or a pre-filled syringe. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for a siRNA compound preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.

This invention is further illustrated by the following examples which should not be construed as limiting. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the informal Sequence Listing and Figures, are hereby incorporated herein by reference.

EXAMPLES Example 1. iRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

siRNA Design

siRNAs targeting the xanthine dehydrogenase (XDH) gene, (human: NCBI refseqID NM_000379; NCBI GeneID: 7498) were designed using custom R and Python scripts. The human NM_000379 REFSEQ mRNA, version 4, has a length of 5715 bases. Detailed lists of the unmodified XDH sense and antisense strand nucleotide sequences are shown in Table 2. Detailed lists of the modified XDH sense and antisense strand nucleotide sequences are shown in Table 3.

It is to be understood that, throughout the application, a duplex name without a decimal is equivalent to a duplex name with a decimal which merely references the batch number of the duplex. For example, AD-959917 is equivalent to AD-959917.1.

siRNA Synthesis

siRNAs were synthesized and annealed using routine methods known in the art.

Briefly, siRNA sequences were synthesized on a 1 μmol scale using a Mermade 192 synthesizer (BioAutomation) with phosphoramidite chemistry on solid supports. The solid support was controlled pore glass (500-1000 Å) loaded with a custom GalNAc ligand (3′-GalNAc conjugates), universal solid support (AM Chemicals), or the first nucleotide of interest. Ancillary synthesis reagents and standard 2-cyanoethyl phosphoramidite monomers (2′-deoxy-2′-fluoro, 2′-O-methyl, RNA, DNA) were obtained from Thermo-Fisher (Milwaukee, Wis.), Hongene (China), or Chemgenes (Wilmington, Mass., USA). Additional phosphoramidite monomers were procured from commercial suppliers, prepared in-house, or procured using custom synthesis from various CMOs. Phosphoramidites were prepared at a concentration of 100 mM in either acetonitrile or 9:1 acetonitrile:DMF and were coupled using 5-Ethylthio-1H-tetrazole (ETT, 0.25 M in acetonitrile) with a reaction time of 400 s. Phosphorothioate linkages were generated using a 100 mM solution of 3-((Dimethylamino-methylidene) amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, Mass., USA)) in anhydrous acetonitrile/pyridine (9:1 v/v). Oxidation time was 5 minutes. All sequences were synthesized with final removal of the DMT group (“DMT-Off”).

Upon completion of the solid phase synthesis, solid-supported oligoribonucleotides were treated with 300 μL of Methylamine (40% aqueous) at room temperature in 96 well plates for approximately 2 hours to afford cleavage from the solid support and subsequent removal of all additional base-labile protecting groups. For sequences containing any natural ribonucleotide linkages (2′-OH) protected with a tert-butyl dimethyl silyl (TBDMS) group, a second deprotection step was performed using TEA.3HF (triethylamine trihydrofluoride). To each oligonucleotide solution in aqueous methylamine was added 200 μL of dimethyl sulfoxide (DMSO) and 300 μL TEA.3HF and the solution was incubated for approximately 30 mins at 60° C. After incubation, the plate was allowed to come to room temperature and crude oligonucleotides were precipitated by the addition of 1 mL of 9:1 acetontrile:ethanol or 1:1 ethanol:isopropanol. The plates were then centrifuged at 4° C. for 45 mins and the supernatant carefully decanted with the aid of a multichannel pipette. The oligonucleotide pellet was resuspended in 20 mM NaOAc and subsequently desalted using a HiTrap size exclusion column (5 mL, GE Healthcare) on an Agilent LC system equipped with an autosampler, UV detector, conductivity meter, and fraction collector. Desalted samples were collected in 96 well plates and then analyzed by LC-MS and UV spectrometry to confirm identity and quantify the amount of material, respectively.

Duplexing of single strands was performed on a Tecan liquid handling robot. Sense and antisense single strands were combined in an equimolar ratio to a final concentration of 10 μM in 1×PBS in 96 well plates, the plate sealed, incubated at 100° C. for 10 minutes, and subsequently allowed to return slowly to room temperature over a period of 2-3 hours. The concentration and identity of each duplex was confirmed and then subsequently utilized for in vitro screening assays.

Example 2. In Vitro Screening Methods

Cell culture and Free Uptake Experiments

Primary mouse hepatocytes (PMH) or primary human hepatocytes (PHH) were freshly isolated less than 1 hour prior to transfection and grown in primary hepatocyte media.

Free uptake experiments were performed by adding 2.5 μl of siRNA duplexes in PBS per well into a 96 well plate. Complete growth media (47.5 μl) containing about 1.5×10⁴ PMH or PHH was then added to the siRNA. Cells were incubated for 48 hours prior to RNA purification and RT-qPCR. Single dose experiments in PHH were performed at 500 nM, 100 nM, 10 nM and/or 0.1 nM in PHH and in PMH at 500 nM, 100 nM, nM, and 1 nM final duplex concentration.

Total RNA isolation was performed using DYNABEADS. Briefly, cells were lysed in 10 μl of Lysis/Binding Buffer containing 3 μL of beads per well and mixed for 10 minutes on an electrostatic shaker. The washing steps were automated on a Biotek EL406, using a magnetic plate support. Beads were washed (in 3 μL) once in Buffer A, once in Buffer B, and twice in Buffer E, with aspiration steps in between. Following a final aspiration, complete 12 μL RT mixture was added to each well, as described below.

For cDNA synthesis, a master mix of 1.5 μl 10× Buffer, 0.6 μl 10×dNTPs, 1.5 μl Random primers, 0.75 μl Reverse Transcriptase, 0.75 μl RNase inhibitor and 9.9 μl of H₂O per reaction were added per well. Plates were sealed, agitated for 10 minutes on an electrostatic shaker, and then incubated at 37 degrees C. for 2 hours. Following this, the plates were agitated at 80 degrees C. for 8 minutes.

For RT-qPCR, two microlitre (μ1) of cDNA were added to a master mix containing 0.5 μl of human GAPDH TaqMan Probe (4326317E), 0.5 μl human APOC3, 41 nuclease-free water and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche).

To calculate relative fold change, data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or mock-transfected. The sense and antisense sequences of AD-1955 are: sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 21) and antisense UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 22).

The results of the free uptake experiments of the dsRNA agents listed in Tables 2 and 3 in PHH are shown in Table 4 and the results of the free uptake experiments of the dsRNA agents listed in Tables 2 and 3 in PMH are shown in Table 5.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds; and it is understood that when the nucleotide contains a 2′-fluoro modification, then the fluoro replaces the hydroxy at that position in the parent nucleotide (i.e., it is a 2′-deoxy-2′-fluoronucleotide). Abbreviation Nucleotide(s) A Adenosine-3′-phosphate Ab beta-L-adenosine-3′-phosphate Abs beta-L-adenosine-3′-phosphorothioate Af 2′-fluoroadenosine-3′-phosphate Afs 2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioate C cytidine-3′-phosphate Cb beta-L-cytidine-3′-phosphate Cbs beta-L-cytidine-3′-phosphorothioate Cf 2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate G guanosine-3′-phosphate Gb beta-L-guanosine-3′-phosphate Gbs beta-L-guanosine-3′-phosphorothioate Gf 2′-fluoroguanosine-3′-phosphate Gfs 2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioate T 5′-methyluridine-3′-phosphate Tf 2′-fluoro-5-methyluridine-3′-phosphate Tfs 2′-fluoro-5-methyluridine-3′-phosphorothioate Ts 5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf 2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine -3′-phosphorothioate Us uridine -3′-phosphorothioate N any nucleotide, modified or unmodified a 2′-O-methyladenosine-3′-phosphate as 2′-O-methyladenosine-3′- phosphorothioate c 2′-O-methylcytidine-3′-phosphate cs 2′-O-methylcytidine-3′- phosphorothioate g 2′-O-methylguanosine-3′-phosphate gs 2′-O-methylguanosine-3′- phosphorothioate t 2′-O-methyl-5-methyluridine-3′-phosphate ts 2′-O-methyl-5-methyluridine-3′-phosphorothioate u 2′-O-methyluridine-3′-phosphate us 2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L10 N-(cholesterylcarboxamidocaproyl)-4-hydroxyprolinol (Hyp-C6-Chol) L96 N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol (Hyp-(GalNAc-alkyl)3)

Y34 2-hydroxymethyl-tetrahydrofurane-4-methoxy-3-phosphate (abasic 2′-OMe furanose) Y44 inverted abasic DNA (2-hydroxymethyl-tetrahydrofurane-5-phosphate) (Agn) Adenosine-glycol nucleic acid (GNA) (Cgn) Cytidine-glycol nucleic acid (GNA) (Ggn) Guanosine-glycol nucleic acid (GNA) (Tgn) Thymidine-glycol nucleic acid (GNA) S-Isomer P Phosphate VP Vinyl-phosphonate dA 2′-deoxyadenosine-3′-phosphate dAs 2′-deoxyadenosine-3′-phosphorothioate dC 2′-deoxycytidine-3′-phosphate dCs 2′-deoxycytidine-3′-phosphorothioate dG 2′-deoxyguanosine-3′-phosphate dGs 2′-deoxyguanosine-3′-phosphorothioate dT 2′- deoxythimidine -3′-phosphate dTs 2′- deoxythimidine -3′-phosphorothioate dU 2′-deoxyuridine dUs 2′-deoxyuridine-3′-phosphorothioate (C2p) cytidine-2′-phosphate (G2p) guanosine-2′-phosphate (U2p) uridine-2′-phosphate (A2p) adenosine-2′-phosphate (Ahd) 2′-O-hexadecyl-adenosine-3′-phosphate (Chd) 2′-O-hexadecyl-cytidine-3′-phosphate (Ghd) 2′-O-hexadecyl-guanosine-3′-phosphate (Uhd) 2′-O-hexadecyl-uridine-3′-phosphate (Aams) 2′-O-(N-methylacetamide)adenosine-3′-phosphorothioate (Gams) 2′-O-(N-methylacetamide)guanosine-3′-phosphorothioate (Tams) 2′-O-(N-methylacetamide)thymidine-3′-phosphorothioate

TABLE 2 Unmodified Sense and Antisense Strand Sequences of Xanthine Dehydrogenase dsRNA Agents SEQ SEQ ID Range in ID Range in Duplex Name Sense Sequence 5′ to 3′ NO: NM_000379.4 Antisense Sequence 5′ to 3′ NO: NM_000379.4 AD-1135979.1 UACCUGCCAGUGUCUCUUAGU 23 17-37 ACUAAGAGACACUGGCAGGUAGU 382 15-37 AD-1135980.1 ACCUGCCAGUGUCUCUUAGGU 24 18-38 ACCUAAGAGACACUGGCAGGUAG 383 16-38 AD-1135981.1 CCUGCCAGUGUCUCUUAGGAU 25 19-39 AUCCUAAGAGACACUGGCAGGUA 384 17-39 AD-1135982.1 UGCCAGUGUCUCUUAGGAGUU 26 21-41 AACUCCUAAGAGACACUGGCAGG 385 19-41 AD-1135983.1 GCCAGUGUCUCUUAGGAGUGU 27 22-42 ACACTCCUAAGAGACACUGGCAG 386 20-42 AD-1135984.1 CCAGUGUCUCUUAGGAGUGAU 28 23-43 AUCACUCCUAAGAGACACUGGCA 387 21-43 AD-1135985.1 CAGUGUCUCUUAGGAGUGAGU 29 24-44 ACUCACUCCUAAGAGACACUGGC 388 22-44 AD-1135986.1 AGUGUCUCUUAGGAGUGAGGU 30 25-45 ACCUCACUCCUAAGAGACACUGG 389 23-45 AD-1135987.1 UGGAGAAAAAUGCAGAUCCAU 31 123-143 AUGGAUCUGCAUUUUUCUCCACC 390 121-143 AD-1135988.1 CCAGAGACAACCCUUUUGGCU 32 140-160 AGCCAAAAGGGUUGUCUCUGGAU 391 138-160 AD-1135989.1 AGAGACAACCCUUUUGGCCUU 33 142-162 AAGGCCAAAAGGGUUGUCUCUGG 392 140-162 AD-1135990.1 GCACAGUGAUGCUCUCCAAGU 34 228-248 ACUUGGAGAGCAUCACUGUGCAA 393 226-248 AD-1135991.1 CACAGUGAUGCUCUCCAAGUU 35 229-249 AACUTGGAGAGCAUCACUGUGCA 394 227-249 AD-1135992.1 GAUGCUCUCCAAGUAUGAUCU 36 235-255 AGAUCAUACUUGGAGAGCAUCAC 395 233-255 AD-1135993.1 AUGCUCUCCAAGUAUGAUCGU 37 236-256 ACGAUCAUACUUGGAGAGCAUCA 396 234-256 AD-1135994.1 UGCUCUCCAAGUAUGAUCGUU 38 237-257 AACGAUCAUACUUGGAGAGCAUC 397 235-257 AD-1135995.1 GCUCUCCAAGUAUGAUCGUCU 39 238-258 AGACGAUCAUACUUGGAGAGCAU 398 236-258 AD-1135996.1 CUCUCCAAGUAUGAUCGUCUU 40 239-259 AAGACGAUCAUACUUGGAGAGCA 399 237-259 AD-1135997.1 GAUCGUCUGCAGAACAAGAUU 41 251-271 AAUCTUGUUCUGCAGACGAUCAU 400 249-271 AD-1135998.1 CGUCUGCAGAACAAGAUCGUU 42 254-274 AACGAUCUUGUUCUGCAGACGAU 401 252-274 AD-1135999.1 GUCUGCAGAACAAGAUCGUCU 43 255-275 AGACGAUCUUGUUCUGCAGACGA 402 253-275 AD-1136000.1 UCUGCAGAACAAGAUCGUCCU 44 256-276 AGGACGAUCUUGUUCUGCAGACG 403 254-276 AD-1136001.1 GCAGAACAAGAUCGUCCACUU 45 259-279 AAGUGGACGAUCUUGUUCUGCAG 404 257-279 AD-1136002.1 CAGAACAAGAUCGUCCACUUU 46 260-280 AAAGTGGACGAUCUUGUUCUGCA 405 258-280 AD-1136003.1 GAACAAGAUCGUCCACUUUUU 47 262-282 AAAAAGTGGACGAUCUUGUUCUG 406 260-282 AD-1136004.1 AACAAGAUCGUCCACUUUUCU 48 263-283 AGAAAAGUGGACGAUCUUGUUCU 407 261-283 AD-1136005.1 ACAAGAUCGUCCACUUUUCUU 49 264-284 AAGAAAAGUGGACGAUCUUGUUC 408 262-284 AD-1136006.1 CAAGAUCGUCCACUUUUCUGU 50 265-285 ACAGAAAAGUGGACGAUCUUGUU 409 263-285 AD-1136007.1 AAGAUCGUCCACUUUUCUGCU 51 266-286 AGCAGAAAAGUGGACGAUCUUGU 410 264-286 AD-1136008.1 CGUCCACUUUUCUGCCAAUGU 52 271-291 ACAUTGGCAGAAAAGUGGACGAU 411 269-291 AD-1136009.1 GUCCACUUUUCUGCCAAUGCU 53 272-292 AGCATUGGCAGAAAAGUGGACGA 412 270-292 AD-1136010.1 UGCUCCUUGCACCAUGUUGCU 54 308-328 AGCAACAUGGUGCAAGGAGCAGA 413 306-328 AD-1136011.1 UCCUUGCACCAUGUUGCAGUU 55 311-331 AACUGCAACAUGGUGCAAGGAGC 414 309-331 AD-1136012.1 UUGCACCAUGUUGCAGUGACU 56 314-334 AGUCACTGCAACAUGGUGCAAGG 415 312-334 AD-1136013.1 CACCAUGUUGCAGUGACAACU 57 317-337 AGUUGUCACUGCAACAUGGUGCA 416 315-337 AD-1136014.1 ACCAUGUUGCAGUGACAACUU 58 318-338 AAGUTGTCACUGCAACAUGGUGC 417 316-338 AD-1136015.1 AGGAGAGAAUUGCCAAAAGCU 59 381-401 AGCUTUTGGCAAUUCUCUCCUGC 418 379-401 AD-1136016.1 GGAGAGAAUUGCCAAAAGCCU 60 382-402 AGGCTUTUGGCAAUUCUCUCCUG 419 380-402 AD-1136017.1 UGGCAUCGUCAUGAGUAUGUU 61 430-450 AACAUACUCAUGACGAUGCCAGG 420 428-450 AD-1136018.1 GGCAUCGUCAUGAGUAUGUAU 62 431-451 AUACAUACUCAUGACGAUGCCAG 421 429-451 AD-1136019.1 GCAUCGUCAUGAGUAUGUACU 63 432-452 AGUACAUACUCAUGACGAUGCCA 422 430-452 AD-1136020.1 CAUCGUCAUGAGUAUGUACAU 64 433-453 AUGUACAUACUCAUGACGAUGCC 423 431-453 AD-1136021.1 AUCGUCAUGAGUAUGUACACU 65 434-454 AGUGTACAUACUCAUGACGAUGC 424 432-454 AD-1136022.1 CGUCAUGAGUAUGUACACACU 66 436-456 AGUGTGTACAUACUCAUGACGAU 425 434-456 AD-1136023.1 GUCAUGAGUAUGUACACACUU 67 437-457 AAGUGUGUACAUACUCAUGACGA 426 435-457 AD-1136024.1 UCAUGAGUAUGUACACACUGU 68 438-458 ACAGTGTGUACAUACUCAUGACG 427 436-458 AD-1136025.1 AAUGCCUUCCAAGGAAAUCUU 69 497-517 AAGATUTCCUUGGAAGGCAUUCU 428 495-517 AD-1136026.1 AUGCCUUCCAAGGAAAUCUGU 70 498-518 ACAGAUUUCCUUGGAAGGCAUUC 429 496-518 AD-1136027.1 UGCCUUCCAAGGAAAUCUGUU 71 499-519 AACAGAUUUCCUUGGAAGGCAUU 430 497-519 AD-1136028.1 GCCUUCCAAGGAAAUCUGUGU 72 500-520 ACACAGAUUUCCUUGGAAGGCAU 431 498-520 AD-1136029.1 CCUUCCAAGGAAAUCUGUGCU 73 501-521 AGCACAGAUUUCCUUGGAAGGCA 432 499-521 AD-1136030.1 GAGAUGAAGUUCAAGAAUAUU 74 875-895 AAUATUCUUGAACUUCAUCUCAA 433 873-895 AD-1136031.1 GAUGAAGUUCAAGAAUAUGCU 75 877-897 AGCAUAUUCUUGAACUUCAUCUC 434 875-897 AD-1136032.1 AAGUUCAAGAAUAUGCUGUUU 76 881-901 AAACAGCAUAUUCUUGAACUUCA 435 879-901 AD-1136033.1 AGUUCAAGAAUAUGCUGUUUU 77 882-902 AAAACAGCAUAUUCUUGAACUUC 436 880-902 AD-1136034.1 GUUCAAGAAUAUGCUGUUUCU 78 883-903 AGAAACAGCAUAUUCUUGAACUU 437 881-903 AD-1136035.1 UUCAAGAAUAUGCUGUUUCCU 79 884-904 AGGAAACAGCAUAUUCUUGAACU 438 882-904 AD-1136036.1 AAGAAUAUGCUGUUUCCUAUU 80 887-907 AAUAGGAAACAGCAUAUUCUUGA 439 885-907 AD-1136037.1 GGGAGUAUUUCUCAGCAUUCU 81 1320-1340 AGAATGCUGAGAAAUACUCCCCC 440 1318-1340 AD-1136038.1 GGAGUAUUUCUCAGCAUUCAU 82 1321-1341 AUGAAUGCUGAGAAAUACUCCCC 441 1319-1341 AD-1136039.1 GAGUAUUUCUCAGCAUUCAAU 83 1322-1342 AUUGAAUGCUGAGAAAUACUCCC 442 1320-1342 AD-1136040.1 AGUAUUUCUCAGCAUUCAAGU 84 1323-1343 ACUUGAAUGCUGAGAAAUACUCC 443 1321-1343 AD-1136041.1 GUAUUUCUCAGCAUUCAAGCU 85 1324-1344 AGCUTGAAUGCUGAGAAAUACUC 444 1322-1344 AD-1136042.1 GUGGCAUGAGAGUUUUAUUCU 86 1383-1403 AGAAUAAAACUCUCAUGCCACUG 445 1381-1403 AD-1136043.1 GGCAUGAGAGUUUUAUUCAAU 87 1385-1405 AUUGAAUAAAACUCUCAUGCCAC 446 1383-1405 AD-1136044.1 CAUGAGAGUUUUAUUCAAGCU 88 1387-1407 AGCUTGAAUAAAACUCUCAUGCC 447 1385-1407 AD-1136045.1 CCUCACCCUCAGCUUCUUCUU 89 1606-1626 AAGAAGAAGCUGAGGGUGAGGGU 448 1604-1626 AD-1136046.1 UCACCCUCAGCUUCUUCUUCU 90 1608-1628 AGAAGAAGAAGCUGAGGGUGAGG 449 1606-1628 AD-1136047.1 UCUUCAAGUUCUACCUGACAU 91 1623-1643 AUGUCAGGUAGAACUUGAAGAAG 450 1621-1643 AD-1136048.1 UUUCGCCAGUGCAACUUUACU 92 1702-1722 AGUAAAGUUGCACUGGCGAAAGU 451 1700-1722 AD-1136049.1 UUCGCCAGUGCAACUUUACUU 93 1703-1723 AAGUAAAGUUGCACUGGCGAAAG 452 1701-1723 AD-1136050.1 UUCCAGGGUUUGUUUGUUUCU 94 1962-1982 AGAAACAAACAAACCCUGGAACC 453 1960-1982 AD-1136051.1 CAGGGUUUGUUUGUUUCAUUU 95 1965-1985 AAAUGAAACAAACAAACCCUGGA 454 1963-1985 AD-1136052.1 GGGUUUGUUUGUUUCAUUUCU 96 1967-1987 AGAAAUGAAACAAACAAACCCUG 455 1965-1987 AD-1136053.1 GGUUUGUUUGUUUCAUUUCCU 97 1968-1988 AGGAAAUGAAACAAACAAACCCU 456 1966-1988 AD-1136054.1 UUGUUUGUUUCAUUUCCGCUU 98 1971-1991 AAGCGGAAAUGAAACAAACAAAC 457 1969-1991 AD-1136055.1 UUCCUGGGAGUAACAUAACUU 99 1998-2018 AAGUUAUGUUACUCCCAGGAACA 458 1996-2018 AD-1136056.1 GGGAGUAACAUAACUGGAAUU 100 2003-2023 AAUUCCAGUUAUGUUACUCCCAG 459 2001-2023 AD-1136057.1 UAACAUAACUGGAAUUUGUAU 101 2008-2028 AUACAAAUUCCAGUUAUGUUACU 460 2006-2028 AD-1136058.1 AACAUAACUGGAAUUUGUAAU 102 2009-2029 AUUACAAAUUCCAGUUAUGUUAC 461 2007-2029 AD-1136059.1 CGAAGGAUAAGGUUACUUGUU 103 2046-2066 AACAAGUAACCUUAUCCUUCGCA 462 2044-2066 AD-1136060.1 GAAGGAUAAGGUUACUUGUGU 104 2047-2067 ACACAAGUAACCUUAUCCUUCGC 463 2045-2067 AD-1136061.1 GGGUGAAAAUCACCUAUGAAU 105 2130-2150 AUUCAUAGGUGAUUUUCACCCCU 464 2128-2150 AD-1136062.1 AAUCACCUAUGAAGAACUACU 106 2137-2157 AGUAGUTCUUCAUAGGUGAUUUU 465 2135-2157 AD-1136063.1 AUCACCUAUGAAGAACUACCU 107 2138-2158 AGGUAGTUCUUCAUAGGUGAUUU 466 2136-2158 AD-1136064.1 UACCAGCCAUUAUCACAAUUU 108 2154-2174 AAAUTGTGAUAAUGGCUGGUAGU 467 2152-2174 AD-1136065.1 GAACAACUCCUUUUAUGGACU 109 2188-2208 AGUCCAUAAAAGGAGUUGUUCUU 468 2186-2208 AD-1136066.1 GGCCAAGAGCACUUCUACCUU 110 2291-2311 AAGGTAGAAGUGCUCUUGGCCAC 469 2289-2311 AD-1136067.1 GAGCACUUCUACCUGGAGACU 111 2297-2317 AGUCTCCAGGUAGAAGUGCUCUU 470 2295-2317 AD-1136068.1 GCACUUCUACCUGGAGACUCU 112 2299-2319 AGAGTCTCCAGGUAGAAGUGCUC 471 2297-2319 AD-1136069.1 CACUUCUACCUGGAGACUCAU 113 2300-2320 AUGAGUCUCCAGGUAGAAGUGCU 472 2298-2320 AD-1136070.1 UCACUGCACCAUUGCUGUUCU 114 2317-2337 AGAACAGCAAUGGUGCAGUGAGU 473 2315-2337 AD-1136071.1 CACUGCACCAUUGCUGUUCCU 115 2318-2338 AGGAACAGCAAUGGUGCAGUGAG 474 2316-2338 AD-1136072.1 CCAUUGCUGUUCCAAAAGGCU 116 2325-2345 AGCCUUUUGGAACAGCAAUGGUG 475 2323-2345 AD-1136073.1 AGCUCUUUGUGUCUACACAGU 117 2361-2381 ACUGTGTAGACACAAAGAGCUCC 476 2359-2381 AD-1136074.1 GCUCUUUGUGUCUACACAGAU 118 2362-2382 AUCUGUGUAGACACAAAGAGCUC 477 2360-2382 AD-1136075.1 CUCUUUGUGUCUACACAGAAU 119 2363-2383 AUUCTGTGUAGACACAAAGAGCU 478 2361-2383 AD-1136076.1 UCUUUGUGUCUACACAGAACU 120 2364-2384 AGUUCUGUGUAGACACAAAGAGC 479 2362-2384 AD-1136077.1 CUUUGUGUCUACACAGAACAU 121 2365-2385 AUGUTCTGUGUAGACACAAAGAG 480 2363-2385 AD-1136078.1 UUUGUGUCUACACAGAACACU 122 2366-2386 AGUGTUCUGUGUAGACACAAAGA 481 2364-2386 AD-1136079.1 ACACAGAACACCAUGAAGACU 123 2375-2395 AGUCTUCAUGGUGUUCUGUGUAG 482 2373-2395 AD-1136080.1 GAGCUUUGUUGCAAAAAUGUU 124 2398-2418 AACAUUUUUGCAACAAAGCUCUG 483 2396-2418 AD-1136081.1 AGCUUUGUUGCAAAAAUGUUU 125 2399-2419 AAACAUUUUUGCAACAAAGCUCU 484 2397-2419 AD-1136082.1 AGGAUCUCUCUCAGAGUAUUU 126 2691-2711 AAAUACTCUGAGAGAGAUCCUGG 485 2689-2711 AD-1136083.1 GGAUCUCUCUCAGAGUAUUAU 127 2692-2712 AUAATACUCUGAGAGAGAUCCUG 486 2690-2712 AD-1136084.1 GAUCUCUCUCAGAGUAUUAUU 128 2693-2713 AAUAAUACUCUGAGAGAGAUCCU 487 2691-2713 AD-1136085.1 AUCUCUCUCAGAGUAUUAUGU 129 2694-2714 ACAUAAUACUCUGAGAGAGAUCC 488 2692-2714 AD-1136086.1 UCUCUCUCAGAGUAUUAUGGU 130 2695-2715 ACCAUAAUACUCUGAGAGAGAUC 489 2693-2715 AD-1136087.1 CUCUCUCAGAGUAUUAUGGAU 131 2696-2716 AUCCAUAAUACUCUGAGAGAGAU 490 2694-2716 AD-1136088.1 UCUCUCAGAGUAUUAUGGAAU 132 2697-2717 AUUCCAUAAUACUCUGAGAGAGA 491 2695-2717 AD-1136089.1 CUCUCAGAGUAUUAUGGAACU 133 2698-2718 AGUUCCAUAAUACUCUGAGAGAG 492 2696-2718 AD-1136090.1 UCUCAGAGUAUUAUGGAACGU 134 2699-2719 ACGUUCCAUAAUACUCUGAGAGA 493 2697-2719 AD-1136091.1 UCAGAGUAUUAUGGAACGAGU 135 2701-2721 ACUCGUUCCAUAAUACUCUGAGA 494 2699-2721 AD-1136092.1 CAGAGUAUUAUGGAACGAGCU 136 2702-2722 AGCUCGUUCCAUAAUACUCUGAG 495 2700-2722 AD-1136093.1 GCUGUGCAAAACCAACCUUCU 137 2776-2796 AGAAGGTUGGUUUUGCACAGCCG 496 2774-2796 AD-1136094.1 CUGUGCAAAACCAACCUUCCU 138 2777-2797 AGGAAGGUUGGUUUUGCACAGCC 497 2775-2797 AD-1136095.1 CCUGACACACUUCAACCAGAU 139 2932-2952 AUCUGGTUGAAGUGUGUCAGGUC 498 2930-2952 AD-1136096.1 UGACACACUUCAACCAGAAGU 140 2934-2954 ACUUCUGGUUGAAGUGUGUCAGG 499 2932-2954 AD-1136097.1 GACACACUUCAACCAGAAGCU 141 2935-2955 AGCUTCTGGUUGAAGUGUGUCAG 500 2933-2955 AD-1136098.1 ACACUUCAACCAGAAGCUUGU 142 2938-2958 ACAAGCUUCUGGUUGAAGUGUGU 501 2936-2958 AD-1136099.1 AUGCCUAGCAAGCUCUCAGUU 143 2989-3009 AACUGAGAGCUUGCUAGGCAUUC 502 2987-3009 AD-1136100.1 CCUAGCAAGCUCUCAGUAUCU 144 2992-3012 AGAUACTGAGAGCUUGCUAGGCA 503 2990-3012 AD-1136101.1 CUAGCAAGCUCUCAGUAUCAU 145 2993-3013 AUGATACUGAGAGCUUGCUAGGC 504 2991-3013 AD-1136102.1 UAGCAAGCUCUCAGUAUCAUU 146 2994-3014 AAUGAUACUGAGAGCUUGCUAGG 505 2992-3014 AD-1136103.1 AGCAAGCUCUCAGUAUCAUGU 147 2995-3015 ACAUGAUACUGAGAGCUUGCUAG 506 2993-3015 AD-1136104.1 CAAGCUCUCAGUAUCAUGCUU 148 2997-3017 AAGCAUGAUACUGAGAGCUUGCU 507 2995-3017 AD-1136105.1 CUCGGAAGAGUGAGGUUGACU 149 3015-3035 AGUCAACCUCACUCUUCCGAGCA 508 3013-3035 AD-1136106.1 AAGGAGAAUUGUUGGAAAAAU 150 3044-3064 AUUUTUCCAACAAUUCUCCUUGU 509 3042-3064 AD-1136107.1 CACCAAGUUUGGAAUAAGCUU 151 3085-3105 AAGCUUAUUCCAAACUUGGUGGG 510 3083-3105 AD-1136108.1 ACCAAGUUUGGAAUAAGCUUU 152 3089-3109 AAAGCUUAUUCCAAACUUGGUGG 511 3087-3109 AD-1136109.1 AGUUCCUUUUCUGAAUCAGGU 153 3109-3129 ACCUGAUUCAGAAAAGGAACUGU 512 3107-3129 AD-1136110.1 GUUCCUUUUCUGAAUCAGGCU 154 3110-3130 AGCCTGAUUCAGAAAAGGAACUG 513 3108-3130 AD-1136111.1 UUCCUUUUCUGAAUCAGGCAU 155 3111-3131 AUGCCUGAUUCAGAAAAGGAACU 514 3109-3131 AD-1136112.1 UACUUCAUGUGUACACAGAUU 156 3138-3158 AAUCTGTGUACACAUGAAGUAGG 515 3136-3158 AD-1136114.1 CAAGGCCUUCAUACCAAAAUU 157 3197-3217 AAUUTUGGUAUGAAGGCCUUGGC 516 3195-3217 AD-1136115.1 AAGGCCUUCAUACCAAAAUGU 158 3198-3218 ACAUUUUGGUAUGAAGGCCUUGG 517 3196-3218 AD-1136116.1 GCCUUCAUACCAAAAUGGUCU 159 3201-3221 AGACCAUUUUGGUAUGAAGGCCU 518 3199-3221 AD-1136117.1 CACCUCUAAGAUUUAUAUCAU 160 3250-3270 AUGAUAUAAAUCUUAGAGGUGGG 519 3248-3270 AD-1136118.1 ACCUCUAAGAUUUAUAUCAGU 161 3251-3271 ACUGAUAUAAAUCUUAGAGGUGG 520 3249-3271 AD-1136119.1 GCGAGACAAGCACUAACACUU 162 3270-3290 AAGUGUTAGUGCUUGUCUCGCUG 521 3268-3290 AD-1136120.1 CGAGACAAGCACUAACACUGU 163 3271-3291 ACAGTGTUAGUGCUUGUCUCGCU 522 3269-3291 AD-1136121.1 GAGACAAGCACUAACACUGUU 164 3272-3292 AACAGUGUUAGUGCUUGUCUCGC 523 3270-3292 AD-1136122.1 AGACAAGCACUAACACUGUGU 165 3273-3293 ACACAGTGUUAGUGCUUGUCUCG 524 3271-3293 AD-1136123.1 CGGCUUGUCAGACCAUCUUGU 166 3354-3374 ACAAGAUGGUCUGACAAGCCGCA 525 3352-3374 AD-1136124.1 GGCUUGUCAGACCAUCUUGAU 167 3355-3375 AUCAAGAUGGUCUGACAAGCCGC 526 3353-3375 AD-1136125.1 GCUUGUCAGACCAUCUUGAAU 168 3356-3376 AUUCAAGAUGGUCUGACAAGCCG 527 3354-3376 AD-1136126.1 CUUGUCAGACCAUCUUGAAAU 169 3357-3377 AUUUCAAGAUGGUCUGACAAGCC 528 3355-3377 AD-1136127.1 UUGUCAGACCAUCUUGAAAAU 170 3358-3378 AUUUTCAAGAUGGUCUGACAAGC 529 3356-3378 AD-1136128.1 UGUCAGACCAUCUUGAAAAGU 171 3359-3379 ACUUUUCAAGAUGGUCUGACAAG 530 3357-3379 AD-1136129.1 GUCAGACCAUCUUGAAAAGGU 172 3360-3380 ACCUUUUCAAGAUGGUCUGACAA 531 3358-3380 AD-1136130.1 UCAGACCAUCUUGAAAAGGCU 173 3361-3381 AGCCUUUUCAAGAUGGUCUGACA 532 3359-3381 AD-1136131.1 ACCCUACAAGAAGAAGAAUCU 174 3385-3405 AGAUTCTUCUUCUUGUAGGGUUC 533 3383-3405 AD-1136132.1 CUGCCACUGGGUUUUAUAGAU 175 3462-3482 AUCUAUAAAACCCAGUGGCAGAC 534 3460-3482 AD-1136133.1 UGCCACUGGGUUUUAUAGAAU 176 3463-3483 AUUCUAUAAAACCCAGUGGCAGA 535 3461-3483 AD-1136134.1 CACUGGGUUUUAUAGAACACU 177 3466-3486 AGUGTUCUAUAAAACCCAGUGGC 536 3464-3486 AD-1136135.1 ACUGGGUUUUAUAGAACACCU 178 3467-3487 AGGUGUTCUAUAAAACCCAGUGG 537 3465-3487 AD-1136136.1 GGGUUUUAUAGAACACCCAAU 179 3470-3490 AUUGGGTGUUCUAUAAAACCCAG 538 3468-3490 AD-1136137.1 GGUUUUAUAGAACACCCAAUU 180 3471-3491 AAUUGGGUGUUCUAUAAAACCCA 539 3469-3491 AD-1136138.1 UGGGCUACAGCUUUGAGACUU 181 3492-3512 AAGUCUCAAAGCUGUAGCCCAGA 540 3490-3512 AD-1136139.1 GGGCUACAGCUUUGAGACUAU 182 3493-3513 AUAGTCTCAAAGCUGUAGCCCAG 541 3491-3513 AD-1136140.1 GCUACAGCUUUGAGACUAACU 183 3495-3515 AGUUAGTCUCAAAGCUGUAGCCC 542 3493-3515 AD-1136141.1 CUACAGCUUUGAGACUAACUU 184 3496-3516 AAGUTAGUCUCAAAGCUGUAGCC 543 3494-3516 AD-1136142.1 UACAGCUUUGAGACUAACUCU 185 3497-3517 AGAGTUAGUCUCAAAGCUGUAGC 544 3495-3517 AD-1136143.1 ACAGCUUUGAGACUAACUCAU 186 3498-3518 AUGAGUTAGUCUCAAAGCUGUAG 545 3496-3518 AD-1136144.1 CAGCUUUGAGACUAACUCAGU 187 3499-3519 ACUGAGUUAGUCUCAAAGCUGUA 546 3497-3519 AD-1136145.1 AGCUUUGAGACUAACUCAGGU 188 3500-3520 ACCUGAGUUAGUCUCAAAGCUGU 547 3498-3520 AD-1136146.1 CUUCCACUACUUCAGCUAUGU 189 3526-3546 ACAUAGCUGAAGUAGUGGAAGGG 548 3524-3546 AD-1136147.1 UUCCACUACUUCAGCUAUGGU 190 3527-3547 ACCAUAGCUGAAGUAGUGGAAGG 549 3525-3547 AD-1136148.1 GUGGCUUGCUCUGAAGUAGAU 191 3548-3568 AUCUACTUCAGAGCAAGCCACCC 550 3546-3568 AD-1136149.1 UGGCUUGCUCUGAAGUAGAAU 192 3549-3569 AUUCTACUUCAGAGCAAGCCACC 551 3547-3569 AD-1136150.1 GGCUUGCUCUGAAGUAGAAAU 193 3550-3570 AUUUCUACUUCAGAGCAAGCCAC 552 3548-3570 AD-1136151.1 GCUUGCUCUGAAGUAGAAAUU 194 3551-3571 AAUUTCTACUUCAGAGCAAGCCA 553 3549-3571 AD-1136152.1 CCUAACAGGAGAUCAUAAGAU 195 3577-3597 AUCUUAUGAUCUCCUGUUAGGCA 554 3575-3597 AD-1136153.1 CUAACAGGAGAUCAUAAGAAU 196 3578-3598 AUUCTUAUGAUCUCCUGUUAGGC 555 3576-3598 AD-1136154.1 UAACAGGAGAUCAUAAGAACU 197 3579-3599 AGUUCUUAUGAUCUCCUGUUAGG 556 3577-3599 AD-1136155.1 AACAGGAGAUCAUAAGAACCU 198 3580-3600 AGGUTCTUAUGAUCUCCUGUUAG 557 3578-3600 AD-1136156.1 CCUCCGCACAGAUAUUGUCAU 199 3598-3618 AUGACAAUAUCUGUGCGGAGGUU 558 3596-3618 AD-1136157.1 CUCCGCACAGAUAUUGUCAUU 200 3599-3619 AAUGACAAUAUCUGUGCGGAGGU 559 3597-3619 AD-1136158.1 UUGGCUCCAGUCUAAACCCUU 201 3624-3644 AAGGGUTUAGACUGGAGCCAACA 560 3622-3644 AD-1136159.1 UUCCUGGCUGCUUCUAUCUUU 202 3872-3892 AAAGAUAGAAGCAGCCAGGAAGA 561 3870-3892 AD-1136160.1 UCCUGGCUGCUUCUAUCUUCU 203 3873-3893 AGAAGAUAGAAGCAGCCAGGAAG 562 3871-3893 AD-1136161.1 CCUGGCUGCUUCUAUCUUCUU 204 3874-3894 AAGAAGAUAGAAGCAGCCAGGAA 563 3872-3894 AD-1136162.1 CUGGCUGCUUCUAUCUUCUUU 205 3875-3895 AAAGAAGAUAGAAGCAGCCAGGA 564 3873-3895 AD-1136163.1 UGGCUGCUUCUAUCUUCUUUU 206 3876-3896 AAAAGAAGAUAGAAGCAGCCAGG 565 3874-3896 AD-1136164.1 GCUGCUUCUAUCUUCUUUGCU 207 3878-3898 AGCAAAGAAGAUAGAAGCAGCCA 566 3876-3898 AD-1136165.1 CUGCUUCUAUCUUCUUUGCCU 208 3879-3899 AGGCAAAGAAGAUAGAAGCAGCC 567 3877-3899 AD-1136166.1 UGCUUCUAUCUUCUUUGCCAU 209 3880-3900 AUGGCAAAGAAGAUAGAAGCAGC 568 3878-3900 AD-1136167.1 GCUUCUAUCUUCUUUGCCAUU 210 3881-3901 AAUGGCAAAGAAGAUAGAAGCAG 569 3879-3901 AD-1136168.1 CUUCUAUCUUCUUUGCCAUCU 211 3882-3902 AGAUGGCAAAGAAGAUAGAAGCA 570 3880-3902 AD-1136169.1 UUCUAUCUUCUUUGCCAUCAU 212 3883-3903 AUGATGGCAAAGAAGAUAGAAGC 571 3881-3903 AD-1136170.1 UCUAUCUUCUUUGCCAUCAAU 213 3884-3904 AUUGAUGGCAAAGAAGAUAGAAG 572 3882-3904 AD-1136171.1 CUAUCUUCUUUGCCAUCAAAU 214 3885-3905 AUUUGATGGCAAAGAAGAUAGAA 573 3883-3905 AD-1136172.1 UAUCUUCUUUGCCAUCAAAGU 215 3886-3906 ACUUUGAUGGCAAAGAAGAUAGA 574 3884-3906 AD-1136173.1 AUCUUCUUUGCCAUCAAAGAU 216 3887-3907 AUCUTUGAUGGCAAAGAAGAUAG 575 3885-3907 AD-1136174.1 CUUCUUUGCCAUCAAAGAUGU 217 3889-3909 ACAUCUUUGAUGGCAAAGAAGAU 576 3887-3909 AD-1136175.1 GAGCUCAGCACACAGGUAAUU 218 3924-3944 AAUUACCUGUGUGCUGAGCUCGA 577 3922-3944 AD-1136176.1 CUCAGCACACAGGUAAUAACU 219 3927-3947 AGUUAUUACCUGUGUGCUGAGCU 578 3925-3947 AD-1136177.1 UCAGCACACAGGUAAUAACGU 220 3928-3948 ACGUUAUUACCUGUGUGCUGAGC 579 3926-3948 AD-1136178.1 CAGCACACAGGUAAUAACGUU 221 3929-3949 AACGUUAUUACCUGUGUGCUGAG 580 3927-3949 AD-1136179.1 ACAGGUAAUAACGUGAAGGAU 222 3935-3955 AUCCTUCACGUUAUUACCUGUGU 581 3933-3955 AD-1136180.1 CAGGUAAUAACGUGAAGGAAU 223 3936-3956 AUUCCUTCACGUUAUUACCUGUG 582 3934-3956 AD-1136181.1 AGGUAAUAACGUGAAGGAACU 224 3937-3957 AGUUCCTUCACGUUAUUACCUGU 583 3935-3957 AD-1136182.1 AUAACGUGAAGGAACUCUUCU 225 3942-3962 AGAAGAGUUCCUUCACGUUAUUA 584 3940-3962 AD-1136183.1 UAACGUGAAGGAACUCUUCCU 226 3943-3963 AGGAAGAGUUCCUUCACGUUAUU 585 3941-3963 AD-1136184.1 CCCAGAAAACUGCAAACCCUU 227 4042-4062 AAGGGUTUGCAGUUUUCUGGGAC 586 4040-4062 AD-1136185.1 CACAGAACAUGGAUCUAUUAU 228 4145-4165 AUAATAGAUCCAUGUUCUGUGGU 587 4143-4165 AD-1136186.1 ACAGAACAUGGAUCUAUUAAU 229 4146-4166 AUUAAUAGAUCCAUGUUCUGUGG 588 4144-4166 AD-1136187.1 CAGAACAUGGAUCUAUUAAAU 230 4147-4167 AUUUAAUAGAUCCAUGUUCUGUG 589 4145-4167 AD-1136188.1 AGAACAUGGAUCUAUUAAAGU 231 4148-4168 ACUUUAAUAGAUCCAUGUUCUGU 590 4146-4168 AD-1136189.1 GAACAUGGAUCUAUUAAAGUU 232 4149-4169 AACUUUAAUAGAUCCAUGUUCUG 591 4147-4169 AD-1136190.1 AACAUGGAUCUAUUAAAGUCU 233 4150-4170 AGACUUUAAUAGAUCCAUGUUCU 592 4148-4170 AD-1136191.1 ACAUGGAUCUAUUAAAGUCAU 234 4151-4171 AUGACUTUAAUAGAUCCAUGUUC 593 4149-4171 AD-1136192.1 CAUGGAUCUAUUAAAGUCACU 235 4152-4172 AGUGACTUUAAUAGAUCCAUGUU 594 4150-4172 AD-1136193.1 AUGGAUCUAUUAAAGUCACAU 236 4153-4173 AUGUGACUUUAAUAGAUCCAUGU 595 4151-4173 AD-1136194.1 UGGAUCUAUUAAAGUCACAGU 237 4154-4174 ACUGTGACUUUAAUAGAUCCAUG 596 4152-4174 AD-1136195.1 GGAUCUAUUAAAGUCACAGAU 238 4155-4175 AUCUGUGACUUUAAUAGAUCCAU 597 4153-4175 AD-1136196.1 GAUCUAUUAAAGUCACAGAAU 239 4156-4176 AUUCTGTGACUUUAAUAGAUCCA 598 4154-4176 AD-1136197.1 ACAAUGAUAAGCAAAUUCAAU 240 4261-4281 AUUGAAUUUGCUUAUCAUUGUGU 599 4259-4281 AD-1136198.1 CAAUGAUAAGCAAAUUCAAAU 241 4262-4282 AUUUGAAUUUGCUUAUCAUUGUG 600 4260-4282 AD-1136199.1 AUGAUAAGCAAAUUCAAAACU 242 4264-4284 AGUUTUGAAUUUGCUUAUCAUUG 601 4262-4284 AD-1136200.1 AUGCCUAAAUGGUGAAUAUGU 243 4288-4308 ACAUAUUCACCAUUUAGGCAUAA 602 4286-4308 AD-1136201.1 UGCCUAAAUGGUGAAUAUGCU 244 4289-4309 AGCAUAUUCACCAUUUAGGCAUA 603 4287-4309 AD-1136202.1 GCCUAAAUGGUGAAUAUGCAU 245 4290-4310 AUGCAUAUUCACCAUUUAGGCAU 604 4288-4310 AD-1136203.1 CCUAAAUGGUGAAUAUGCAAU 246 4291-4311 AUUGCATAUUCACCAUUUAGGCA 605 4289-4311 AD-1136204.1 CUAAAUGGUGAAUAUGCAAUU 247 4292-4312 AAUUGCAUAUUCACCAUUUAGGC 606 4290-4312 AD-1136205.1 AAUGGUGAAUAUGCAAUUAGU 248 4295-4315 ACUAAUUGCAUAUUCACCAUUUA 607 4293-4315 AD-1136206.1 CGGGAAGGGUUUGUGCUAUUU 249 4357-4377 AAAUAGCACAAACCCUUCCCGAC 608 4355-4377 AD-1136207.1 GGGAAGGGUUUGUGCUAUUCU 250 4358-4378 AGAATAGCACAAACCCUUCCCGA 609 4356-4378 AD-1136208.1 GGAAGGGUUUGUGCUAUUCCU 251 4359-4379 AGGAAUAGCACAAACCCUUCCCG 610 4357-4379 AD-1136209.1 GUAUAACCUCAAGUUCUGAUU 252 4397-4417 AAUCAGAACUUGAGGUUAUACAG 611 4395-4417 AD-1136210.1 UAUAACCUCAAGUUCUGAUGU 253 4398-4418 ACAUCAGAACUUGAGGUUAUACA 612 4396-4418 AD-1136211.1 CCUCAAGUUCUGAUGGUGUCU 254 4403-4423 AGACACCAUCAGAACUUGAGGUU 613 4401-4423 AD-1136212.1 CAAGUUCUGAUGGUGUCUGUU 255 4406-4426 AACAGACACCAUCAGAACUUGAG 614 4404-4426 AD-1136213.1 CCACAAACCUCUAGAAGCUUU 256 4442-4462 AAAGCUTCUAGAGGUUUGUGGGA 615 4440-4462 AD-1136214.1 ACAAACCUCUAGAAGCUUAAU 257 4444-4464 AUUAAGCUUCUAGAGGUUUGUGG 616 4442-4464 AD-1136215.1 AAACCUCUAGAAGCUUAAACU 258 4446-4466 AGUUUAAGCUUCUAGAGGUUUGU 617 4444-4466 AD-1136216.1 AACCUCUAGAAGCUUAAACCU 259 4447-4467 AGGUUUAAGCUUCUAGAGGUUUG 618 4445-4467 AD-1136217.1 UGGCCUUCAAACCAAUGAACU 260 4504-4524 AGUUCAUUGGUUUGAAGGCCAGG 619 4502-4524 AD-1136218.1 GGCCUUCAAACCAAUGAACAU 261 4505-4525 AUGUTCAUUGGUUUGAAGGCCAG 620 4503-4525 AD-1136219.1 GCCUUCAAACCAAUGAACAGU 262 4506-4526 ACUGUUCAUUGGUUUGAAGGCCA 621 4504-4526 AD-1136220.1 UCAAACCAAUGAACAGCAAAU 263 4510-4530 AUUUGCTGUUCAUUGGUUUGAAG 622 4508-4530 AD-1136221.1 GAACAGCAAAGCAUAACCUUU 264 4520-4540 AAAGGUUAUGCUUUGCUGUUCAU 623 4518-4540 AD-1136222.1 AACAGCAAAGCAUAACCUUGU 265 4521-4541 ACAAGGTUAUGCUUUGCUGUUCA 624 4519-4541 AD-1136223.1 AGCAAAGCAUAACCUUGAAUU 266 4524-4544 AAUUCAAGGUUAUGCUUUGCUGU 625 4522-4544 AD-1136224.1 GCAAAGCAUAACCUUGAAUCU 267 4525-4545 AGAUTCAAGGUUAUGCUUUGCUG 626 4523-4545 AD-1136225.1 CAAAGCAUAACCUUGAAUCUU 268 4526-4546 AAGATUCAAGGUUAUGCUUUGCU 627 4524-4546 AD-1136226.1 GCAUAACCUUGAAUCUAUACU 269 4530-4550 AGUAUAGAUUCAAGGUUAUGCUU 628 4528-4550 AD-1136227.1 CAUAACCUUGAAUCUAUACUU 270 4531-4551 AAGUAUAGAUUCAAGGUUAUGCU 629 4529-4551 AD-1136228.1 AUAACCUUGAAUCUAUACUCU 271 4532-4552 AGAGUAUAGAUUCAAGGUUAUGC 630 4530-4552 AD-1136229.1 UAACCUUGAAUCUAUACUCAU 272 4533-4553 AUGAGUAUAGAUUCAAGGUUAUG 631 4531-4553 AD-1136230.1 AACCUUGAAUCUAUACUCAAU 273 4534-4554 AUUGAGTAUAGAUUCAAGGUUAU 632 4532-4554 AD-1136231.1 ACCUUGAAUCUAUACUCAAAU 274 4535-4555 AUUUGAGUAUAGAUUCAAGGUUA 633 4533-4555 AD-1136232.1 CCUUGAAUCUAUACUCAAAUU 275 4536-4556 AAUUTGAGUAUAGAUUCAAGGUU 634 4534-4556 AD-1136233.1 AAUCUAUACUCAAAUUUUGCU 276 4541-4561 AGCAAAAUUUGAGUAUAGAUUCA 635 4539-4561 AD-1136234.1 AUCUAUACUCAAAUUUUGCAU 277 4542-4562 AUGCAAAAUUUGAGUAUAGAUUC 636 4540-4562 AD-1136235.1 GGUUAAAUCCUCUAACCAUCU 278 4579-4599 AGAUGGTUAGAGGAUUUAACCUU 637 4577-4599 AD-1136236.1 UAAAUCCUCUAACCAUCUUUU 279 4582-4602 AAAAGATGGUUAGAGGAUUUAAC 638 4580-4602 AD-1136237.1 AAAUCCUCUAACCAUCUUUGU 280 4583-4603 ACAAAGAUGGUUAGAGGAUUUAA 639 4581-4603 AD-1136238.1 AAUCCUCUAACCAUCUUUGAU 281 4584-4604 AUCAAAGAUGGUUAGAGGAUUUA 640 4582-4604 AD-1136239.1 AUCCUCUAACCAUCUUUGAAU 282 4585-4605 AUUCAAAGAUGGUUAGAGGAUUU 641 4583-4605 AD-1136240.1 UCCUCUAACCAUCUUUGAAUU 283 4586-4606 AAUUCAAAGAUGGUUAGAGGAUU 642 4584-4606 AD-1136241.1 CCUCUAACCAUCUUUGAAUCU 284 4587-4607 AGAUTCAAAGAUGGUUAGAGGAU 643 4585-4607 AD-1136242.1 CUCUAACCAUCUUUGAAUCAU 285 4588-4608 AUGATUCAAAGAUGGUUAGAGGA 644 4586-4608 AD-1136243.1 UCUAACCAUCUUUGAAUCAUU 286 4589-4609 AAUGAUUCAAAGAUGGUUAGAGG 645 4587-4609 AD-1136244.1 CUAACCAUCUUUGAAUCAUUU 287 4590-4610 AAAUGATUCAAAGAUGGUUAGAG 646 4588-4610 AD-1136245.1 UAACCAUCUUUGAAUCAUUGU 288 4591-4611 ACAATGAUUCAAAGAUGGUUAGA 647 4589-4611 AD-1136246.1 AACCAUCUUUGAAUCAUUGGU 289 4592-4612 ACCAAUGAUUCAAAGAUGGUUAG 648 4590-4612 AD-1136247.1 CCAUCUUUGAAUCAUUGGAAU 290 4594-4614 AUUCCAAUGAUUCAAAGAUGGUU 649 4592-4614 AD-1136248.1 CAUCUUUGAAUCAUUGGAAAU 291 4595-4615 AUUUCCAAUGAUUCAAAGAUGGU 650 4593-4615 AD-1136249.1 AUCUUUGAAUCAUUGGAAAGU 292 4596-4616 ACUUTCCAAUGAUUCAAAGAUGG 651 4594-4616 AD-1136250.1 CUUUGAAUCAUUGGAAAGAAU 293 4598-4618 AUUCTUTCCAAUGAUUCAAAGAU 652 4596-4618 AD-1136251.1 GAAAGAAUAAAGAAUGAAACU 294 4611-4631 AGUUTCAUUCUUUAUUCUUUCCA 653 4609-4631 AD-1136252.1 AAAGAAUAAAGAAUGAAACAU 295 4612-4632 AUGUTUCAUUCUUUAUUCUUUCC 654 4610-4632 AD-1136253.1 GAAUGAAACAAAUUCAAGGUU 296 4622-4642 AACCUUGAAUUUGUUUCAUUCUU 655 4620-4642 AD-1136254.1 AUGAAACAAAUUCAAGGUUAU 297 4624-4644 AUAACCTUGAAUUUGUUUCAUUC 656 4622-4644 AD-1136255.1 AACAAAUUCAAGGUUAAUUGU 298 4628-4648 ACAAUUAACCUUGAAUUUGUUUC 657 4626-4648 AD-1136256.1 UGAAGCUGCAUAAAGCAAGAU 299 4660-4680 AUCUTGCUUUAUGCAGCUUCACA 658 4658-4680 AD-1136257.1 AAGCUGCAUAAAGCAAGAUUU 300 4662-4682 AAAUCUTGCUUUAUGCAGCUUCA 659 4660-4682 AD-1136258.1 AGCUGCAUAAAGCAAGAUUAU 301 4663-4683 AUAATCTUGCUUUAUGCAGCUUC 660 4661-4683 AD-1136259.1 GCUGCAUAAAGCAAGAUUACU 302 4664-4684 AGUAAUCUUGCUUUAUGCAGCUU 661 4662-4684 AD-1136260.1 CUGCAUAAAGCAAGAUUACUU 303 4665-4685 AAGUAAUCUUGCUUUAUGCAGCU 662 4663-4685 AD-1136261.1 UGCAUAAAGCAAGAUUACUCU 304 4666-4686 AGAGUAAUCUUGCUUUAUGCAGC 663 4664-4686 AD-1136262.1 CAUAAAGCAAGAUUACUCUAU 305 4668-4688 AUAGAGTAAUCUUGCUUUAUGCA 664 4666-4688 AD-1136263.1 UAAAGCAAGAUUACUCUAUAU 306 4670-4690 AUAUAGAGUAAUCUUGCUUUAUG 665 4668-4690 AD-1136264.1 AUACAAAAAUCCAACCAACUU 307 4690-4710 AAGUTGGUUGGAUUUUUGUAUUA 666 4688-4710 AD-1136265.1 UACAAAAAUCCAACCAACUCU 308 4691-4711 AGAGTUGGUUGGAUUUUUGUAUU 667 4689-4711 AD-1136266.1 ACAAAAAUCCAACCAACUCAU 309 4692-4712 AUGAGUTGGUUGGAUUUUUGUAU 668 4690-4712 AD-1136267.1 CAAAAAUCCAACCAACUCAAU 310 4693-4713 AUUGAGTUGGUUGGAUUUUUGUA 669 4691-4713 AD-1136268.1 AAAAAUCCAACCAACUCAAUU 311 4694-4714 AAUUGAGUUGGUUGGAUUUUUGU 670 4692-4714 AD-1136269.1 AAAAUCCAACCAACUCAAUUU 312 4695-4715 AAAUTGAGUUGGUUGGAUUUUUG 671 4693-4715 AD-1136270.1 AAAUCCAACCAACUCAAUUAU 313 4696-4716 AUAATUGAGUUGGUUGGAUUUUU 672 4694-4716 AD-1136271.1 AAUCCAACCAACUCAAUUAUU 314 4697-4717 AAUAAUUGAGUUGGUUGGAUUUU 673 4695-4717 AD-1136272.1 AUCCAACCAACUCAAUUAUUU 315 4698-4718 AAAUAAUUGAGUUGGUUGGAUUU 674 4696-4718 AD-1136273.1 UCCAACCAACUCAAUUAUUGU 316 4699-4719 ACAAUAAUUGAGUUGGUUGGAUU 675 4697-4719 AD-1136274.1 CCAACCAACUCAAUUAUUGAU 317 4700-4720 AUCAAUAAUUGAGUUGGUUGGAU 676 4698-4720 AD-1136275.1 CAACCAACUCAAUUAUUGAGU 318 4701-4721 ACUCAAUAAUUGAGUUGGUUGGA 677 4699-4721 AD-1136276.1 AACCAACUCAAUUAUUGAGCU 319 4702-4722 AGCUCAAUAAUUGAGUUGGUUGG 678 4700-4722 AD-1136277.1 ACCAACUCAAUUAUUGAGCAU 320 4703-4723 AUGCTCAAUAAUUGAGUUGGUUG 679 4701-4723 AD-1136278.1 CCAACUCAAUUAUUGAGCACU 321 4704-4724 AGUGCUCAAUAAUUGAGUUGGUU 680 4702-4724 AD-1136279.1 CGUACAAUGUUCUAGAUUUCU 322 4723-4743 AGAAAUCUAGAACAUUGUACGUG 681 4721-4743 AD-1136280.1 GUACAAUGUUCUAGAUUUCUU 323 4724-4744 AAGAAAUCUAGAACAUUGUACGU 682 4722-4744 AD-1136281.1 UACAAUGUUCUAGAUUUCUUU 324 4725-4745 AAAGAAAUCUAGAACAUUGUACG 683 4723-4745 AD-1136282.1 ACAAUGUUCUAGAUUUCUUUU 325 4726-4746 AAAAGAAAUCUAGAACAUUGUAC 684 4724-4746 AD-1136283.1 CAAUGUUCUAGAUUUCUUUCU 326 4727-4747 AGAAAGAAAUCUAGAACAUUGUA 685 4725-4747 AD-1136284.1 AAUGUUCUAGAUUUCUUUCCU 327 4728-4748 AGGAAAGAAAUCUAGAACAUUGU 686 4726-4748 AD-1136285.1 GUUCUAGAUUUCUUUCCCUUU 328 4731-4751 AAAGGGAAAGAAAUCUAGAACAU 687 4729-4751 AD-1136286.1 UUCUAGAUUUCUUUCCCUUCU 329 4732-4752 AGAAGGGAAAGAAAUCUAGAACA 688 4730-4752 AD-1136287.1 UCUUUACAUUUGAAGCCAAAU 330 4817-4837 AUUUGGCUUCAAAUGUAAAGAUU 689 4815-4837 AD-1136288.1 CUUUACAUUUGAAGCCAAAGU 331 4818-4838 ACUUTGGCUUCAAAUGUAAAGAU 690 4816-4838 AD-1136289.1 UUUACAUUUGAAGCCAAAGUU 332 4819-4839 AACUTUGGCUUCAAAUGUAAAGA 691 4817-4839 AD-1136290.1 ACAUUUGAAGCCAAAGUAAUU 333 4822-4842 AAUUACTUUGGCUUCAAAUGUAA 692 4820-4842 AD-1136291.1 AUUUGAAGCCAAAGUAAUUUU 334 4824-4844 AAAAUUACUUUGGCUUCAAAUGU 693 4822-4844 AD-1136292.1 GAAGCCAAAGUAAUUUCCACU 335 4828-4848 AGUGGAAAUUACUUUGGCUUCAA 694 4826-4848 AD-1136293.1 AAGCCAAAGUAAUUUCCACCU 336 4829-4849 AGGUGGAAAUUACUUUGGCUUCA 695 4827-4849 AD-1136294.1 CCAAAGUAAUUUCCACCUAGU 337 4832-4852 ACUAGGTGGAAAUUACUUUGGCU 696 4830-4852 AD-1136295.1 CAAAGUAAUUUCCACCUAGAU 338 4833-4853 AUCUAGGUGGAAAUUACUUUGGC 697 4831-4853 AD-1136296.1 AAAGUAAUUUCCACCUAGAAU 339 4834-4854 AUUCTAGGUGGAAAUUACUUUGG 698 4832-4854 AD-1136297.1 GCAAGAUCUUGUCUCUGAAGU 340 5134-5154 ACUUCAGAGACAAGAUCUUGCUC 699 5132-5154 AD-1136298.1 AGGUAGAAGAGCCAAGAAGCU 341 5201-5221 AGCUTCTUGGCUCUUCUACCUCU 700 5199-5221 AD-1136299.1 CUAGCUCUGUCUCUUCUGUCU 342 5234-5254 AGACAGAAGAGACAGAGCUAGGA 701 5232-5254 AD-1136300.1 UAGCUCUGUCUCUUCUGUCUU 343 5235-5255 AAGACAGAAGAGACAGAGCUAGG 702 5233-5255 AD-1136301.1 AGCUCUGUCUCUUCUGUCUCU 344 5236-5256 AGAGACAGAAGAGACAGAGCUAG 703 5234-5256 AD-1136302.1 AUCUUUGUGAUCUUGGACUGU 345 5257-5277 ACAGUCCAAGAUCACAAAGAUAG 704 5255-5277 AD-1136303.1 CUUCCUGUGAUCCAUUUUACU 346 5286-5306 AGUAAAAUGGAUCACAGGAAGGG 705 5284-5306 AD-1136304.1 UUCCUGUGAUCCAUUUUACUU 347 5287-5307 AAGUAAAAUGGAUCACAGGAAGG 706 5285-5307 AD-1136305.1 UCCUGUGAUCCAUUUUACUGU 348 5288-5308 ACAGUAAAAUGGAUCACAGGAAG 707 5286-5308 AD-1136306.1 CCUGUGAUCCAUUUUACUGCU 349 5289-5309 AGCAGUAAAAUGGAUCACAGGAA 708 5287-5309 AD-1136307.1 CUGUGAUCCAUUUUACUGCAU 350 5290-5310 AUGCAGTAAAAUGGAUCACAGGA 709 5288-5310 AD-1136308.1 CUAUAAAUAUAUGACCUGAAU 351 5360-5380 AUUCAGGUCAUAUAUUUAUAGGC 710 5358-5380 AD-1136309.1 UGACCUGAAAACUCCAGUUAU 352 5371-5391 AUAACUGGAGUUUUCAGGUCAUA 711 5369-5391 AD-1136310.1 GACCUGAAAACUCCAGUUACU 353 5372-5392 AGUAACTGGAGUUUUCAGGUCAU 712 5370-5392 AD-1136311.1 AAGGAUCUGCAGCUAUCUAAU 354 5395-5415 AUUAGATAGCUGCAGAUCCUUUA 713 5393-5415 AD-1136312.1 AGGAUCUGCAGCUAUCUAAGU 355 5396-5416 ACUUAGAUAGCUGCAGAUCCUUU 714 5394-5416 AD-1136313.1 GCUAUCUAAGGCUUGGUUUUU 356 5406-5426 AAAAACCAAGCCUUAGAUAGCUG 715 5404-5426 AD-1136314.1 CUAUCUAAGGCUUGGUUUUCU 357 5407-5427 AGAAAACCAAGCCUUAGAUAGCU 716 5405-5427 AD-1136315.1 AUCUAAGGCUUGGUUUUCUUU 358 5409-5429 AAAGAAAACCAAGCCUUAGAUAG 717 5407-5429 AD-1136316.1 UCUAAGGCUUGGUUUUCUUAU 359 5410-5430 AUAAGAAAACCAAGCCUUAGAUA 718 5408-5430 AD-1136317.1 CUAAGGCUUGGUUUUCUUACU 360 5411-5431 AGUAAGAAAACCAAGCCUUAGAU 719 5409-5431 AD-1136318.1 UAAGGCUUGGUUUUCUUACUU 361 5412-5432 AAGUAAGAAAACCAAGCCUUAGA 720 5410-5432 AD-1136319.1 GGCUUGGUUUUCUUACUGUCU 362 5415-5435 AGACAGTAAGAAAACCAAGCCUU 721 5413-5435 AD-1136320.1 GCUUGGUUUUCUUACUGUCAU 363 5416-5436 AUGACAGUAAGAAAACCAAGCCU 722 5414-5436 AD-1136321.1 CUUGGUUUUCUUACUGUCAUU 364 5417-5437 AAUGACAGUAAGAAAACCAAGCC 723 5415-5437 AD-1136322.1 UUGGUUUUCUUACUGUCAUAU 365 5418-5438 AUAUGACAGUAAGAAAACCAAGC 724 5416-5438 AD-1136323.1 UGGUUUUCUUACUGUCAUAUU 366 5419-5439 AAUATGACAGUAAGAAAACCAAG 725 5417-5439 AD-1136324.1 GGUUUUCUUACUGUCAUAUGU 367 5420-5440 ACAUAUGACAGUAAGAAAACCAA 726 5418-5440 AD-1136325.1 UUUUCUUACUGUCAUAUGAUU 368 5422-5442 AAUCAUAUGACAGUAAGAAAACC 727 5420-5442 AD-1136326.1 UUUCUUACUGUCAUAUGAUAU 369 5423-5443 AUAUCAUAUGACAGUAAGAAAAC 728 5421-5443 AD-1136327.1 UCUUACUGUCAUAUGAUACCU 370 5425-5445 AGGUAUCAUAUGACAGUAAGAAA 729 5423-5445 AD-1136328.1 CUUACUGUCAUAUGAUACCUU 371 5426-5446 AAGGUAUCAUAUGACAGUAAGAA 730 5424-5446 AD-1136329.1 UCUGCGGUUGGUAAAGAGAAU 372 5481-5501 AUUCTCTUUACCAACCGCAGAAA 731 5479-5501 AD-1136330.1 UGGUGAUUUAAUCCCUACAUU 373 5537-5557 AAUGTAGGGAUUAAAUCACCAUU 732 5535-5557 AD-1136331.1 GGUGAUUUAAUCCCUACAUGU 374 5538-5558 ACAUGUAGGGAUUAAAUCACCAU 733 5536-5558 AD-1136332.1 UGUAUAAAUCCAACCUUCUGU 375 5597-5617 ACAGAAGGUUGGAUUUAUACAGU 734 5595-5617 AD-1136333.1 GUGCUUGAUGUCACUAAUAAU 376 5679-5699 AUUATUAGUGACAUCAAGCACCA 735 5677-5699 AD-1136334.1 UGCUUGAUGUCACUAAUAAAU 377 5680-5700 AUUUAUUAGUGACAUCAAGCACC 736 5678-5700 AD-1136335.1 GCUUGAUGUCACUAAUAAAUU 378 5681-5701 AAUUUAUUAGUGACAUCAAGCAC 737 5679-5701 AD-1136336.1 AUGUCACUAAUAAAUGAAACU 379 5686-5706 AGUUTCAUUUAUUAGUGACAUCA 738 5684-5706 AD-1136337.1 UGUCACUAAUAAAUGAAACUU 380 5687-5707 AAGUTUCAUUUAUUAGUGACAUC 739 5685-5707 AD-1136338.1 UAAUAAAUGAAACUGUCAGCU 381 5693-5713 AGCUGACAGUUUCAUUUAUUAGU 740 5691-5713

TABLE 3 Modified Sense and Antisense Strand Sequences of Xanthine Dehydrogenase dsRNA Agents SEQ SEQ SEQ Duplex ID ID ID Name Sense Sequence 5′ to 3′ NO: Antisense Sequence 5′ to 3′ NO: mRNA Target Sequence NO: AD- usasccugCfcAfGfUfgucucuua 741 asCfsuaaGfagacacuGfgCfagguasgs 1100 ACUACCUGCCAGUGUCUCUUA 1459 1135979.1 guL96 u GG AD- ascscugcCfaGfUfGfucucuuag 742 asCfscuaAfgagacacUfgGfcaggusas 1101 CUACCUGCCAGUGUCUCUUAG 1460 1135980.1 guL96 g GA AD- cscsugccAfgUfGfUfcucuuagg 743 asUfsccuAfagagacaCfuGfgcaggsus 1102 UACCUGCCAGUGUCUCUUAGG 1461 1135981.1 auL96 a AG AD- usgsccagUfgUfCfUfcuuaggag 744 asAfscucCfuaagagaCfaCfuggcasgs 1103 CCUGCCAGUGUCUCUUAGGAG 1462 1135982.1 uuL96 g UG AD- gscscaguGfuCfUfCfuuaggagu 745 asCfsacdTc(C2p)uaagagAfcAfcugg 1104 CUGCCAGUGUCUCUUAGGAGU 1463 1135983.1 guL96 csasg GA AD- cscsagugUfcUfCfUfuaggagug 746 asUfscadCu(C2p)cuaagaGfaCfacug 1105 UGCCAGUGUCUCUUAGGAGUG 1464 1135984.1 auL96 gscsa AG AD- csasguguCfuCfUfUfaggaguga 747 asCfsucaCfuccuaagAfgAfcacugsgs 1106 GCCAGUGUCUCUUAGGAGUGA 1465 1135985.1 guL96 c GG AD- asgsugucUfcUfUfAfggaguga 748 asCfscucAfcuccuaaGfaGfacacusgs 1107 CCAGUGUCUCUUAGGAGUGAG 1466 1135986.1 gguL96 g GU AD- usgsgagaAfaAfAfUfgcagaucc 749 asUfsggdAu(C2p)ugcauuUfuUfcuc 1108 GGUGGAGAAAAAUGCAGAUC 1467 1135987.1 auL96 cascsc CAG AD- cscsagagAfcAfAfCfccuuuugg 750 asGfsccaAfaaggguuGfuCfucuggsas 1109 AUCCAGAGACAACCCUUUUGG 1468 1135988.1 cuL96 u CC AD- asgsagacAfaCfCfCfuuuuggcc 751 asAfsggdCc(Agn)aaagggUfuGfucu 1110 CCAGAGACAACCCUUUUGGCC 1469 1135989.1 uuL96 cusgsg UA AD- gscsacagUfgAfUfGfcucuccaa 752 asCfsuudGg(Agn)gagcauCfaCfugu 1111 UUGCACAGUGAUGCUCUCCAA 1470 1135990.1 guL96 gcsasa GU AD- csascaguGfaUfGfCfucuccaag 753 asAfscudTg(G2p)agagcaUfcAfcug 1112 UGCACAGUGAUGCUCUCCAAG 1471 1135991.1 uuL96 ugscsa UA AD- gsasugcuCfuCfCfAfaguaugau 754 asGfsaucAfuacuuggAfgAfgcaucsas 1113 GUGAUGCUCUCCAAGUAUGAU 1472 1135992.1 cuL96 c CG AD- asusgcucUfcCfAfAfguaugauc 755 asCfsgauCfauacuugGfaGfagcauscs 1114 UGAUGCUCUCCAAGUAUGAUC 1473 1135993.1 guL96 a GU AD- usgscucuCfcAfAfGfuaugaucg 756 asAfscgaUfcauacuuGfgAfgagcasus 1115 GAUGCUCUCCAAGUAUGAUCG 1474 1135994.1 uuL96 c UC AD- gscsucucCfaAfGfUfaugaucgu 757 asGfsacgAfucauacuUfgGfagagcsas 1116 AUGCUCUCCAAGUAUGAUCGU 1475 1135995.1 cuL96 u CU AD- csuscuccAfaGfUfAfugaucguc 758 asAfsgacGfaucauacUfuGfgagagscs 1117 UGCUCUCCAAGUAUGAUCGUC 1476 1135996.1 uuL96 a UG AD- gsasucguCfuGfCfAfgaacaaga 759 asAfsucdTu(G2p)uucugcAfgAfcga 1118 AUGAUCGUCUGCAGAACAAGA 1477 1135997.1 uuL96 ucsasu UC AD- csgsucugCfaGfAfAfcaagaucg 760 asAfscgaUfcuuguucUfgCfagacgsas 1119 AUCGUCUGCAGAACAAGAUCG 1478 1135998.1 uuL96 u UC AD- gsuscugcAfgAfAfCfaagaucgu 761 asGfsacgAfucuuguuCfuGfcagacsgs 1120 UCGUCUGCAGAACAAGAUCGU 1479 1135999.1 cuL96 a CC AD- uscsugcaGfaAfCfAfagaucguc 762 asGfsgacGfaucuuguUfcUfgcagascs 1121 CGUCUGCAGAACAAGAUCGUC 1480 1136000.1 cuL96 g CA AD- gscsagaaCfaAfGfAfucguccac 763 asAfsgudGg(Agn)cgaucuUfgUfucu 1122 CUGCAGAACAAGAUCGUCCAC 1481 1136001.1 uuL96 gcsasg UU AD- csasgaacAfaGfAfUfcguccacu 764 asAfsagdTg(G2p)acgaucUfuGfuuc 1123 UGCAGAACAAGAUCGUCCACU 1482 1136002.1 uuL96 ugscsa UU AD- gsasacaaGfaUfCfGfuccacuuu 765 asAfsaadAg(Tgn)ggacgaUfcUfugu 1124 CAGAACAAGAUCGUCCACUUU 1483 1136003.1 uuL96 ucsusg UC AD- asascaagAfuCfGfUfccacuuuu 766 asGfsaaaAfguggacgAfuCfuuguuscs 1125 AGAACAAGAUCGUCCACUUUU 1484 1136004.1 cuL96 u CU AD- ascsaagaUfcGfUfCfcacuuuuc 767 asAfsgaaAfaguggacGfaUfcuugusus 1126 GAACAAGAUCGUCCACUUUUC 1485 1136005.1 uuL96 c UG AD- csasagauCfgUfCfCfacuuuucu 768 asCfsagaAfaaguggaCfgAfucuugsus 1127 AACAAGAUCGUCCACUUUUCU 1486 1136006.1 guL96 u GC AD- asasgaucGfuCfCfAfcuuuucug 769 asGfscagAfaaaguggAfcGfaucuusgs 1128 ACAAGAUCGUCCACUUUUCUG 1487 1136007.1 cuL96 u CC AD- csgsuccaCfuUfUfUfcugccaau 770 asCfsaudTg(G2p)cagaaaAfgUfggac 1129 AUCGUCCACUUUUCUGCCAAU 1488 1136008.1 guL96 gsasu GC AD- gsusccacUfuUfUfCfugccaaug 771 asGfscadTu(G2p)gcagaaAfaGfugg 1130 UCGUCCACUUUUCUGCCAAUG 1489 1136009.1 cuL96 acsgsa CC AD- usgscuccUfuGfCfAfccauguug 772 asGfscaaCfauggugcAfaGfgagcasgs 1131 UCUGCUCCUUGCACCAUGUUG 1490 1136010.1 cuL96 a CA AD- uscscuugCfaCfCfAfuguugcag 773 asAfscudGc(Agn)acauggUfgCfaag 1132 GCUCCUUGCACCAUGUUGCAG 1491 1136011.1 uuL96 gasgsc UG AD- ususgcacCfaUfGfUfugcaguga 774 asGfsucdAc(Tgn)gcaacaUfgGfugca 1133 CCUUGCACCAUGUUGCAGUGA 1492 1136012.1 cuL96 asgsg CA AD- csasccauGfuUfGfCfagugacaa 775 asGfsuudGu(C2p)acugcaAfcAfugg 1134 UGCACCAUGUUGCAGUGACAA 1493 1136013.1 cuL96 ugscsa CU AD- ascscaugUfuGfCfAfgugacaac 776 asAfsgudTg(Tgn)cacugcAfaCfaugg 1135 GCACCAUGUUGCAGUGACAAC 1494 1136014.1 uuL96 usgsc UG AD- asgsgagaGfaAfUfUfgccaaaag 777 asGfscudTu(Tgn)ggcaauUfcUfcucc 1136 GCAGGAGAGAAUUGCCAAAA 1495 1136015.1 cuL96 usgsc GCC AD- gsgsagagAfaUfUfGfccaaaagc 778 asGfsgcdTu(Tgn)uggcaaUfuCfucuc 1137 CAGGAGAGAAUUGCCAAAAGC 1496 1136016.1 cuL96 csusg CA AD- usgsgcauCfgUfCfAfugaguau 779 asAfscauAfcucaugaCfgAfugccasgs 1138 CCUGGCAUCGUCAUGAGUAUG 1497 1136017.1 guuL96 g UA AD- gsgscaucGfuCfAfUfgaguaug 780 asUfsacdAu(Agn)cucaugAfcGfaug 1139 CUGGCAUCGUCAUGAGUAUGU 1498 1136018.1 uauL96 ccsasg AC AD- gscsaucgUfcAfUfGfaguaugua 781 asGfsuacAfuacucauGfaCfgaugcscs 1140 UGGCAUCGUCAUGAGUAUGU 1499 1136019.1 cuL96 a ACA AD- csasucguCfaUfGfAfguauguac 782 asUfsgudAc(Agn)uacucaUfgAfcga 1141 GGCAUCGUCAUGAGUAUGUAC 1500 1136020.1 auL96 ugscsc AC AD- asuscgucAfuGfAfGfuauguaca 783 asGfsugdTa(C2p)auacucAfuGfacga 1142 GCAUCGUCAUGAGUAUGUACA 1501 1136021.1 cuL96 usgsc CA AD- csgsucauGfaGfUfAfuguacaca 784 asGfsugdTg(Tgn)acauacUfcAfugac 1143 AUCGUCAUGAGUAUGUACACA 1502 1136022.1 cuL96 gsasu CU AD- gsuscaugAfgUfAfUfguacacac 785 asAfsgudGu(G2p)uacauaCfuCfaug 1144 UCGUCAUGAGUAUGUACACAC 1503 1136023.1 uuL96 acsgsa UG AD- uscsaugaGfuAfUfGfuacacacu 786 asCfsagdTg(Tgn)guacauAfcUfcaug 1145 CGUCAUGAGUAUGUACACACU 1504 1136024.1 guL96 ascsg GC AD- asasugccUfuCfCfAfaggaaauc 787 asAfsgadTu(Tgn)ccuuggAfaGfgca 1146 AGAAUGCCUUCCAAGGAAAUC 1505 1136025.1 uuL96 uuscsu UG AD- asusgccuUfcCfAfAfggaaaucu 788 asCfsagaUfuuccuugGfaAfggcausus 1147 GAAUGCCUUCCAAGGAAAUCU 1506 1136026.1 guL96 c GU AD- usgsccuuCfcAfAfGfgaaaucug 789 asAfscagAfuuuccuuGfgAfaggcasus 1148 AAUGCCUUCCAAGGAAAUCUG 1507 1136027.1 uuL96 u UG AD- gscscuucCfaAfGfGfaaaucugu 790 asCfsacaGfauuuccuUfgGfaaggcsas 1149 AUGCCUUCCAAGGAAAUCUGU 1508 1136028.1 guL96 u GC AD- cscsuuccAfaGfGfAfaaucugug 791 asGfscacAfgauuuccUfuGfgaaggscs 1150 UGCCUUCCAAGGAAAUCUGUG 1509 1136029.1 cuL96 a CC AD- gsasgaugAfaGfUfUfcaagaaua 792 asAfsuadTu(C2p)uugaacUfuCfauc 1151 UUGAGAUGAAGUUCAAGAAU 1510 1136030.1 uuL96 ucsasa AUG AD- gsasugaaGfuUfCfAfagaauaug 793 asGfscauAfuucuugaAfcUfucaucsus 1152 GAGAUGAAGUUCAAGAAUAU 1511 1136031.1 cuL96 c GCU AD- asasguucAfaGfAfAfuaugcug 794 asAfsacdAg(C2p)auauucUfuGfaac 1153 UGAAGUUCAAGAAUAUGCUG 1512 1136032.1 uuuL96 uuscsa UUU AD- asgsuucaAfgAfAfUfaugcugu 795 asAfsaadCa(G2p)cauauuCfuUfgaac 1154 GAAGUUCAAGAAUAUGCUGU 1513 1136033.1 uuuL96 ususc UUC AD- gsusucaaGfaAfUfAfugcuguu 796 asGfsaadAc(Agn)gcauauUfcUfuga 1155 AAGUUCAAGAAUAUGCUGUU 1514 1136034.1 ucuL96 acsusu UCC AD- ususcaagAfaUfAfUfgcuguuu 797 asGfsgaaAfcagcauaUfuCfuugaascs 1156 AGUUCAAGAAUAUGCUGUUU 1515 1136035.1 ccuL96 u CCU AD- asasgaauAfuGfCfUfguuuccua 798 asAfsuadGg(Agn)aacagcAfuAfuuc 1157 UCAAGAAUAUGCUGUUUCCUA 1516 1136036.1 uuL96 uusgsa UG AD- gsgsgaguAfuUfUfCfucagcau 799 asGfsaadTg(C2p)ugagaaAfuAfcucc 1158 GGGGGAGUAUUUCUCAGCAU 1517 1136037.1 ucuL96 cscsc UCA AD- gsgsaguaUfuUfCfUfcagcauuc 800 asUfsgadAu(G2p)cugagaAfaUfacu 1159 GGGGAGUAUUUCUCAGCAUUC 1518 1136038.1 auL96 ccscsc AA AD- gsasguauUfuCfUfCfagcauuca 801 asUfsugaAfugcugagAfaAfuacucscs 1160 GGGAGUAUUUCUCAGCAUUCA 1519 1136039.1 auL96 c AG AD- asgsuauuUfcUfCfAfgcauucaa 802 asCfsuugAfaugcugaGfaAfauacuscs 1161 GGAGUAUUUCUCAGCAUUCAA 1520 1136040.1 guL96 c GC AD- gsusauuuCfuCfAfGfcauucaag 803 asGfscudTg(Agn)augcugAfgAfaau 1162 GAGUAUUUCUCAGCAUUCAAG 1521 1136041.1 cuL96 acsusc CA AD- gsusggcaUfgAfGfAfguuuuau 804 asGfsaauAfaaacucuCfaUfgccacsus 1163 CAGUGGCAUGAGAGUUUUAU 1522 1136042.1 ucuL96 g UCA AD- gsgscaugAfgAfGfUfuuuauuc 805 asUfsugaAfuaaaacuCfuCfaugccsas 1164 GUGGCAUGAGAGUUUUAUUC 1523 1136043.1 aauL96 c AAG AD- csasugagAfgUfUfUfuauucaag 806 asGfscudTg(Agn)auaaaaCfuCfucau 1165 GGCAUGAGAGUUUUAUUCAA 1524 1136044.1 cuL96 gscsc GCC AD- cscsucacCfcUfCfAfgcuucuuc 807 asAfsgaaGfaagcugaGfgGfugaggsgs 1166 ACCCUCACCCUCAGCUUCUUC 1525 1136045.1 uuL96 u UU AD- uscsacccUfcAfGfCfuucuucuu 808 asGfsaadGa(Agn)gaagcuGfaGfggu 1167 CCUCACCCUCAGCUUCUUCUU 1526 1136046.1 cuL96 gasgsg CA AD- uscsuucaAfgUfUfCfuaccugac 809 asUfsgudCa(G2p)guagaaCfuUfgaa 1168 CUUCUUCAAGUUCUACCUGAC 1527 1136047.1 auL96 gasasg AG AD- ususucgcCfaGfUfGfcaacuuua 810 asGfsuaaAfguugcacUfgGfcgaaasgs 1169 ACUUUCGCCAGUGCAACUUUA 1528 1136048.1 cuL96 u CU AD- ususcgccAfgUfGfCfaacuuuac 811 asAfsguaAfaguugcaCfuGfgcgaasas 1170 CUUUCGCCAGUGCAACUUUAC 1529 1136049.1 uuL96 g UG AD- ususccagGfgUfUfUfguuuguu 812 asGfsaaaCfaaacaaaCfcCfuggaascsc 1171 GGUUCCAGGGUUUGUUUGUU 1530 1136050.1 ucuL96 UCA AD-1 csasggguUfuGfUfUfuguuuca 813 asAfsaugAfaacaaacAfaAfcccugsgs 1172 UCCAGGGUUUGUUUGUUUCA 1531 136051.1 uuuL96 a UUU AD- gsgsguuuGfuUfUfGfuuucauu 814 asGfsaaaUfgaaacaaAfcAfaacccsusg 1173 CAGGGUUUGUUUGUUUCAUU 1532 1136052.1 ucuL96 UCC AD- gsgsuuugUfuUfGfUfuucauuu 815 asGfsgaaAfugaaacaAfaCfaaaccscsu 1174 AGGGUUUGUUUGUUUCAUUU 1533 1136053.1 ccuL96 CCG AD- ususguuuGfuUfUfCfauuuccg 816 asAfsgcgGfaaaugaaAfcAfaacaasas 1175 GUUUGUUUGUUUCAUUUCCGC 1534 1136054.1 cuuL96 c UG AD- ususccugGfgAfGfUfaacauaac 817 asAfsguuAfuguuacuCfcCfaggaascs 1176 UGUUCCUGGGAGUAACAUAAC 1535 1136055.1 uuL96 a UG AD- gsgsgaguAfaCfAfUfaacuggaa 818 asAfsuudCc(Agn)guuaugUfuAfcuc 1177 CUGGGAGUAACAUAACUGGA 1536 1136056.1 uuL96 ccsasg AUU AD- usasacauAfaCfUfGfgaauuugu 819 asUfsacaAfauuccagUfuAfuguuascs 1178 AGUAACAUAACUGGAAUUUG 1537 1136057.1 auL96 u UAA AD- asascauaAfcUfGfGfaauuugua 820 asUfsuacAfaauuccaGfuUfauguusas 1179 GUAACAUAACUGGAAUUUGU 1538 1136058.1 auL96 c AAU AD- csgsaaggAfuAfAfGfguuacuu 821 asAfscaaGfuaaccuuAfuCfcuucgscs 1180 UGCGAAGGAUAAGGUUACUU 1539 1136059.1 guuL96 a GUG AD- gsasaggaUfaAfGfGfuuacuug 822 asCfsacaAfguaaccuUfaUfccuucsgs 1181 GCGAAGGAUAAGGUUACUUG 1540 1136060.1 uguL96 c UGU AD- gsgsgugaAfaAfUfCfaccuauga 823 asUfsucaUfaggugauUfuUfcacccscs 1182 AGGGGUGAAAAUCACCUAUG 1541 1136061.1 auL96 u AAG AD- asasucacCfuAfUfGfaagaacua 824 asGfsuadGu(Tgn)cuucauAfgGfuga 1183 AAAAUCACCUAUGAAGAACUA 1542 1136062.1 cuL96 uususu CC AD- asuscaccUfaUfGfAfagaacuac 825 asGfsgudAg(Tgn)ucuucaUfaGfgug 1184 AAAUCACCUAUGAAGAACUAC 1543 1136063.1 cuL96 aususu CA AD- usasccagCfcAfUfUfaucacaau 826 asAfsaudTg(Tgn)gauaauGfgCfugg 1185 ACUACCAGCCAUUAUCACAAU 1544 1136064.1 uuL96 uasgsu UG AD- gsasacaaCfuCfCfUfuuuaugga 827 asGfsuccAfuaaaaggAfgUfuguucsus 1186 AAGAACAACUCCUUUUAUGGA 1545 1136065.1 cuL96 u CC AD- gsgsccaaGfaGfCfAfcuucuacc 828 asAfsggdTa(G2p)aagugcUfcUfugg 1187 GUGGCCAAGAGCACUUCUACC 1546 1136066.1 uuL96 ccsasc UG AD- gsasgcacUfuCfUfAfccuggaga 829 asGfsucdTc(C2p)agguagAfaGfugc 1188 AAGAGCACUUCUACCUGGAGA 1547 1136067.1 cuL96 ucsusu CU AD- gscsacuuCfuAfCfCfuggagacu 830 asGfsagdTc(Tgn)ccagguAfgAfagu 1189 GAGCACUUCUACCUGGAGACU 1548 1136068.1 cuL96 gcsusc CA AD- csascuucUfaCfCfUfggagacuc 831 asUfsgadGu(C2p)uccaggUfaGfaag 1190 AGCACUUCUACCUGGAGACUC 1549 1136069.1 auL96 ugscsu AC AD- uscsacugCfaCfCfAfuugcuguu 832 asGfsaadCa(G2p)caauggUfgCfagu 1191 ACUCACUGCACCAUUGCUGUU 1550 1136070.1 cuL96 gasgsu CC AD- csascugcAfcCfAfUfugcuguuc 833 asGfsgadAc(Agn)gcaaugGfuGfcag 1192 CUCACUGCACCAUUGCUGUUC 1551 1136071.1 cuL96 ugsasg CA AD- cscsauugCfuGfUfUfccaaaagg 834 asGfsccuUfuuggaacAfgCfaauggsus 1193 CACCAUUGCUGUUCCAAAAGG 1552 1136072.1 cuL96 g CG AD- asgscucuUfuGfUfGfucuacaca 835 asCfsugdTg(Tgn)agacacAfaAfgagc 1194 GGAGCUCUUUGUGUCUACACA 1553 1136073.1 guL96 uscsc GA AD- gscsucuuUfgUfGfUfcuacacag 836 asUfscudGu(G2p)uagacaCfaAfaga 1195 GAGCUCUUUGUGUCUACACAG 1554 1136074.1 auL96 gcsusc AA AD- csuscuuuGfuGfUfCfuacacaga 837 asUfsucdTg(Tgn)guagacAfcAfaaga 1196 AGCUCUUUGUGUCUACACAGA 1555 1136075.1 auL96 gscsu AC AD- uscsuuugUfgUfCfUfacacagaa 838 asGfsuudCu(G2p)uguagaCfaCfaaa 1197 GCUCUUUGUGUCUACACAGAA 1556 1136076.1 cuL96 gasgsc CA AD- csusuuguGfuCfUfAfcacagaac 839 asUfsgudTc(Tgn)guguagAfcAfcaaa 1198 CUCUUUGUGUCUACACAGAAC 1557 1136077.1 auL96 gsasg AC AD- ususugugUfcUfAfCfacagaaca 840 asGfsugdTu(C2p)uguguaGfaCfaca 1199 UCUUUGUGUCUACACAGAACA 1558 1136078.1 cuL96 aasgsa CC AD- ascsacagAfaCfAfCfcaugaaga 841 asGfsucdTu(C2p)auggugUfuCfugu 1200 CUACACAGAACACCAUGAAGA 1559 1136079.1 cuL96 gusasg CC AD- gsasgcuuUfgUfUfGfcaaaaaug 842 asAfscauUfuuugcaaCfaAfagcucsus 1201 CAGAGCUUUGUUGCAAAAAU 1560 1136080.1 uuL96 g GUU AD- asgscuuuGfuUfGfCfaaaaaugu 843 asAfsacaUfuuuugcaAfcAfaagcuscs 1202 AGAGCUUUGUUGCAAAAAUG 1561 1136081.1 uuL96 u UUG AD- asgsgaucUfcUfCfUfcagaguau 844 asAfsaudAc(Tgn)cugagaGfaGfaucc 1203 CCAGGAUCUCUCUCAGAGUAU 1562 1136082.1 uuL96 usgsg UA AD- gsgsaucuCfuCfUfCfagaguauu 845 asUfsaadTa(C2p)ucugagAfgAfgau 1204 CAGGAUCUCUCUCAGAGUAUU 1563 1136083.1 auL96 ccsusg AU AD- gsasucucUfcUfCfAfgaguauua 846 asAfsuaaUfacucugaGfaGfagaucscs 1205 AGGAUCUCUCUCAGAGUAUUA 1564 1136084.1 uuL96 u UG AD- asuscucuCfuCfAfGfaguauuau 847 asCfsauaAfuacucugAfgAfgagauscs 1206 GGAUCUCUCUCAGAGUAUUAU 1565 1136085.1 guL96 c GG AD- uscsucucUfcAfGfAfguauuau 848 asCfscauAfauacucuGfaGfagagasus 1207 GAUCUCUCUCAGAGUAUUAUG 1566 1136086.1 gguL96 c GA AD- csuscucuCfaGfAfGfuauuaugg 849 asUfsccaUfaauacucUfgAfgagagsas 1208 AUCUCUCUCAGAGUAUUAUGG 1567 1136087.1 auL96 u AA AD- uscsucucAfgAfGfUfauuaugg 850 asUfsuccAfuaauacuCfuGfagagasgs 1209 UCUCUCUCAGAGUAUUAUGGA 1568 1136088.1 aauL96 a AC AD- csuscucaGfaGfUfAfuuauggaa 851 asGfsuudCc(Agn)uaauacUfcUfgag 1210 CUCUCUCAGAGUAUUAUGGAA 1569 1136089.1 cuL96 agsasg CG AD- uscsucagAfgUfAfUfuauggaac 852 asCfsguuCfcauaauaCfuCfugagasgs 1211 UCUCUCAGAGUAUUAUGGAAC 1570 1136090.1 guL96 a GA AD- uscsagagUfaUfUfAfuggaacga 853 asCfsucgUfuccauaaUfaCfucugasgs 1212 UCUCAGAGUAUUAUGGAACG 1571 1136091.1 guL96 a AGC AD- csasgaguAfuUfAfUfggaacgag 854 asGfscucGfuuccauaAfuAfcucugsas 1213 CUCAGAGUAUUAUGGAACGA 1572 1136092.1 cuL96 g GCU AD- gscsugugCfaAfAfAfccaaccuu 855 asGfsaadGg(Tgn)ugguuuUfgCfaca 1214 CGGCUGUGCAAAACCAACCUU 1573 1136093.1 cuL96 gcscsg CC AD- csusgugcAfaAfAfCfcaaccuuc 856 asGfsgadAg(G2p)uugguuUfuGfcac 1215 GGCUGUGCAAAACCAACCUUC 1574 1136094.1 cuL96 agscsc CC AD- cscsugacAfcAfCfUfucaaccag 857 asUfscudGg(Tgn)ugaaguGfuGfuca 1216 GACCUGACACACUUCAACCAG 1575 1136095.1 auL96 ggsusc AA AD- usgsacacAfcUfUfCfaaccagaa 858 asCfsuudCu(G2p)guugaaGfuGfugu 1217 CCUGACACACUUCAACCAGAA 1576 1136096.1 guL96 casgsg GC AD- gsascacaCfuUfCfAfaccagaag 859 asGfscudTc(Tgn)gguugaAfgUfgug 1218 CUGACACACUUCAACCAGAAG 1577 1136097.1 cuL96 ucsasg CU AD- ascsacuuCfaAfCfCfagaagcuu 860 asCfsaagCfuucugguUfgAfagugusgs 1219 ACACACUUCAACCAGAAGCUU 1578 1136098.1 guL96 u GA AD- asusgccuAfgCfAfAfgcucucag 861 asAfscudGa(G2p)agcuugCfuAfggc 1220 GAAUGCCUAGCAAGCUCUCAG 1579 1136099.1 uuL96 aususc UA AD- cscsuagcAfaGfCfUfcucaguau 862 asGfsaudAc(Tgn)gagagcUfuGfcua 1221 UGCCUAGCAAGCUCUCAGUAU 1580 1136100.1 cuL96 ggscsa CA AD- csusagcaAfgCfUfCfucaguauc 863 asUfsgadTa(C2p)ugagagCfuUfgcu 1222 GCCUAGCAAGCUCUCAGUAUC 1581 1136101.1 auL96 agsgsc AU AD- usasgcaaGfcUfCfUfcaguauca 864 asAfsugaUfacugagaGfcUfugcuasgs 1223 CCUAGCAAGCUCUCAGUAUCA 1582 1136102.1 uuL96 g UG AD- asgscaagCfuCfUfCfaguaucau 865 asCfsaugAfuacugagAfgCfuugcusas 1224 CUAGCAAGCUCUCAGUAUCAU 1583 1136103.1 guL96 g GC AD- csasagcuCfuCfAfGfuaucaugc 866 asAfsgcdAu(G2p)auacugAfgAfgcu 1225 AGCAAGCUCUCAGUAUCAUGC 1584 1136104.1 uuL96 ugscsu UC AD- csuscggaAfgAfGfUfgagguug 867 asGfsucdAa(C2p)cucacuCfuUfccga 1226 UGCUCGGAAGAGUGAGGUUG 1585 1136105.1 acuL96 gscsa ACA AD- asasggagAfaUfUfGfuuggaaaa 868 asUfsuudTu(C2p)caacaaUfuCfuccu 1227 ACAAGGAGAAUUGUUGGAAA 1586 1136106.1 auL96 usgsu AAG AD- csasccaaGfuUfUfGfgaauaagc 869 asAfsgcuUfauuccaaAfcUfuggugsgs 1228 CCCACCAAGUUUGGAAUAAGC 1587 1136107.1 uuL96 g UU AD- ascscaagUfuUfGfGfaauaagcu 870 asAfsagcUfuauuccaAfaCfuuggusgs 1229 CCACCAAGUUUGGAAUAAGCU 1588 1136108.1 uuL96 g UU AD- asgsuuccUfuUfUfCfugaaucag 871 asCfscugAfuucagaaAfaGfgaacusgs 1230 ACAGUUCCUUUUCUGAAUCAG 1589 1136109.1 guL96 u GC AD- gsusuccuUfuUfCfUfgaaucagg 872 asGfsccdTg(Agn)uucagaAfaAfgga 1231 CAGUUCCUUUUCUGAAUCAGG 1590 1136110.1 cuL96 acsusg CA AD- ususccuuUfuCfUfGfaaucaggc 873 asUfsgcdCu(G2p)auucagAfaAfagg 1232 AGUUCCUUUUCUGAAUCAGGC 1591 1136111.1 auL96 aascsu AG AD- usascuucAfuGfUfGfuacacaga 874 asAfsucdTg(Tgn)guacacAfuGfaagu 1233 CCUACUUCAUGUGUACACAGA 1592 1136112.1 uuL96 asgsg UG AD- csasaggcCfuUfCfAfuaccaaaa 875 asAfsuudTu(G2p)guaugaAfgGfccu 1234 GCCAAGGCCUUCAUACCAAAA 1593 1136114.1 uuL96 ugsgsc UG AD- asasggccUfuCfAfUfaccaaaau 876 asCfsauuUfugguaugAfaGfgccuusgs 1235 CCAAGGCCUUCAUACCAAAAU 1594 1136115.1 guL96 g GG AD- gscscuucAfuAfCfCfaaaauggu 877 asGfsaccAfuuuugguAfuGfaaggcscs 1236 AGGCCUUCAUACCAAAAUGGU 1595 1136116.1 cuL96 u CC AD- csasccucUfaAfGfAfuuuauauc 878 asUfsgauAfuaaaucuUfaGfaggugsgs 1237 CCCACCUCUAAGAUUUAUAUC 1596 1136117.1 auL96 g AG AD- ascscucuAfaGfAfUfuuauauca 879 asCfsugaUfauaaaucUfuAfgaggusgs 1238 CCACCUCUAAGAUUUAUAUCA 1597 1136118.1 guL96 g GC AD- gscsgagaCfaAfGfCfacuaacac 880 asAfsgudGu(Tgn)agugcuUfgUfcuc 1239 CAGCGAGACAAGCACUAACAC 1598 1136119.1 uuL96 gcsusg UG AD- csgsagacAfaGfCfAfcuaacacu 881 asCfsagdTg(Tgn)uagugcUfuGfucu 1240 AGCGAGACAAGCACUAACACU 1599 1136120.1 guL96 cgscsu GU AD- gsasgacaAfgCfAfCfuaacacug 882 asAfscadGu(G2p)uuagugCfuUfguc 1241 GCGAGACAAGCACUAACACUG 1600 1136121.1 uuL96 ucsgsc UG AD- asgsacaaGfcAfCfUfaacacugu 883 asCfsacdAg(Tgn)guuaguGfcUfugu 1242 CGAGACAAGCACUAACACUGU 1601 1136122.1 guL96 cuscsg GC AD- csgsgcuuGfuCfAfGfaccaucuu 884 asCfsaagAfuggucugAfcAfagccgscs 1243 UGCGGCUUGUCAGACCAUCUU 1602 1136123.1 guL96 a GA AD- gsgscuugUfcAfGfAfccaucuu 885 asUfscaaGfauggucuGfaCfaagccsgs 1244 GCGGCUUGUCAGACCAUCUUG 1603 1136124.1 gauL96 c AA AD- gscsuuguCfaGfAfCfcaucuuga 886 asUfsucdAa(G2p)auggucUfgAfcaa 1245 CGGCUUGUCAGACCAUCUUGA 1604 1136125.1 auL96 gcscsg AA AD- csusugucAfgAfCfCfaucuugaa 887 asUfsuudCa(Agn)gaugguCfuGfaca 1246 GGCUUGUCAGACCAUCUUGAA 1605 1136126.1 auL96 agscsc AA AD- ususgucaGfaCfCfAfucuugaaa 888 asUfsuudTc(Agn)agauggUfcUfgac 1247 GCUUGUCAGACCAUCUUGAAA 1606 1136127.1 auL96 aasgsc AG AD- usgsucagAfcCfAfUfcuugaaaa 889 asCfsuuuUfcaagaugGfuCfugacasas 1248 CUUGUCAGACCAUCUUGAAAA 1607 1136128.1 guL96 g GG AD- gsuscagaCfcAfUfCfuugaaaag 890 asCfscuuUfucaagauGfgUfcugacsas 1249 UUGUCAGACCAUCUUGAAAAG 1608 1136129.1 guL96 a GC AD- uscsagacCfaUfCfUfugaaaagg 891 asGfsccuUfuucaagaUfgGfucugascs 1250 UGUCAGACCAUCUUGAAAAGG 1609 1136130.1 cuL96 a CU AD- ascsccuaCfaAfGfAfagaagaau 892 asGfsaudTc(Tgn)ucuucuUfgUfagg 1251 GAACCCUACAAGAAGAAGAAU 1610 1136131.1 cuL96 gususc CC AD- csusgccaCfuGfGfGfuuuuauag 893 asUfscuaUfaaaacccAfgUfggcagsas 1252 GUCUGCCACUGGGUUUUAUAG 1611 1136132.1 auL96 c AA AD- usgsccacUfgGfGfUfuuuauaga 894 asUfsucuAfuaaaaccCfaGfuggcasgs 1253 UCUGCCACUGGGUUUUAUAGA 1612 1136133.1 auL96 a AC AD- csascuggGfuUfUfUfauagaaca 895 asGfsugdTu(C2p)uauaaaAfcCfcagu 1254 GCCACUGGGUUUUAUAGAACA 1613 1136134.1 cuL96 gsgsc CC AD- ascsugggUfuUfUfAfuagaacac 896 asGfsgudGu(Tgn)cuauaaAfaCfccag 1255 CCACUGGGUUUUAUAGAACAC 1614 1136135.1 cuL96 usgsg CC AD- gsgsguuuUfaUfAfGfaacaccca 897 asUfsugdGg(Tgn)guucuaUfaAfaac 1256 CUGGGUUUUAUAGAACACCCA 1615 1136136.1 auL96 ccsasg AU AD- gsgsuuuuAfuAfGfAfacacccaa 898 asAfsuudGg(G2p)uguucuAfuAfaaa 1257 UGGGUUUUAUAGAACACCCAA 1616 1136137.1 uuL96 ccscsa UC AD- usgsggcuAfcAfGfCfuuugaga 899 asAfsgudCu(C2p)aaagcuGfuAfgcc 1258 UCUGGGCUACAGCUUUGAGAC 1617 1136138.1 cuuL96 casgsa UA AD-1 gsgsgcuaCfaGfCfUfuugagacu 900 asUfsagdTc(Tgn)caaagcUfgUfagcc 1259 CUGGGCUACAGCUUUGAGACU 1618 1136139. auL96 csasg AA AD- gscsuacaGfcUfUfUfgagacuaa 901 asGfsuudAg(Tgn)cucaaaGfcUfgua 1260 GGGCUACAGCUUUGAGACUAA 1619 1136140.1 cuL96 gcscsc CU AD- csusacagCfuUfUfGfagacuaac 902 asAfsgudTa(G2p)ucucaaAfgCfugu 1261 GGCUACAGCUUUGAGACUAAC 1620 1136141.1 uuL96 agscsc UC AD- usascagcUfuUfGfAfgacuaacu 903 asGfsagdTu(Agn)gucucaAfaGfcug 1262 GCUACAGCUUUGAGACUAACU 1621 1136142.1 cuL96 uasgsc CA AD- ascsagcuUfuGfAfGfacuaacuc 904 asUfsgadGu(Tgn)agucucAfaAfgcu 1263 CUACAGCUUUGAGACUAACUC 1622 1136143.1 auL96 gusasg AG AD- csasgcuuUfgAfGfAfcuaacuca 905 asCfsugaGfuuagucuCfaAfagcugsus 1264 UACAGCUUUGAGACUAACUCA 1623 1136144.1 guL96 a GG AD- asgscuuuGfaGfAfCfuaacucag 906 asCfscudGa(G2p)uuagucUfcAfaag 1265 ACAGCUUUGAGACUAACUCAG 1624 1136145.1 guL96 cusgsu GG AD- csusuccaCfuAfCfUfucagcuau 907 asCfsaudAg(C2p)ugaaguAfgUfgga 1266 CCCUUCCACUACUUCAGCUAU 1625 1136146.1 guL96 agsgsg GG AD- ususccacUfaCfUfUfcagcuaug 908 asCfscauAfgcugaagUfaGfuggaasgs 1267 CCUUCCACUACUUCAGCUAUG 1626 1136147.1 guL96 g GG AD- gsusggcuUfgCfUfCfugaagua 909 asUfscudAc(Tgn)ucagagCfaAfgcca 1268 GGGUGGCUUGCUCUGAAGUA 1627 1136148.1 gauL96 cscsc GAA AD- usgsgcuuGfcUfCfUfgaaguaga 910 asUfsucdTa(C2p)uucagaGfcAfagcc 1269 GGUGGCUUGCUCUGAAGUAG 1628 1136149.1 auL96 ascsc AAA AD- gsgscuugCfuCfUfGfaaguagaa 911 asUfsuudCu(Agn)cuucagAfgCfaag 1270 GUGGCUUGCUCUGAAGUAGA 1629 1136150.1 auL96 ccsasc AAU AD- gscsuugcUfcUfGfAfaguagaaa 912 asAfsuudTc(Tgn)acuucaGfaGfcaag 1271 UGGCUUGCUCUGAAGUAGAA 1630 1136151.1 uuL96 cscsa AUC AD- cscsuaacAfgGfAfGfaucauaag 913 asUfscuuAfugaucucCfuGfuuaggscs 1272 UGCCUAACAGGAGAUCAUAAG 1631 1136152.1 auL96 a AA AD- csusaacaGfgAfGfAfucauaaga 914 asUfsucdTu(Agn)ugaucuCfcUfguu 1273 GCCUAACAGGAGAUCAUAAGA 1632 1136153.1 auL96 agsgsc AC AD- usasacagGfaGfAfUfcauaagaa 915 asGfsuucUfuaugaucUfcCfuguuasgs 1274 CCUAACAGGAGAUCAUAAGAA 1633 1136154.1 cuL96 g CC AD- asascaggAfgAfUfCfauaagaac 916 asGfsgudTc(Tgn)uaugauCfuCfcug 1275 CUAACAGGAGAUCAUAAGAAC 1634 1136155.1 cuL96 uusasg CU AD- cscsuccgCfaCfAfGfauauuguc 917 asUfsgacAfauaucugUfgCfggaggsus 1276 AACCUCCGCACAGAUAUUGUC 1635 1136156.1 auL96 u AU AD- csusccgcAfcAfGfAfuauuguca 918 asAfsugaCfaauaucuGfuGfcggagsgs 1277 ACCUCCGCACAGAUAUUGUCA 1636 1136157.1 uuL96 u UG AD- ususggcuCfcAfGfUfcuaaaccc 919 asAfsggdGu(Tgn)uagacuGfgAfgcc 1278 UGUUGGCUCCAGUCUAAACCC 1637 1136158.1 uuL96 aascsa UG AD- ususccugGfcUfGfCfuucuaucu 920 asAfsagaUfagaagcaGfcCfaggaasgs 1279 UCUUCCUGGCUGCUUCUAUCU 1638 1136159.1 uuL96 a UC AD- uscscuggCfuGfCfUfucuaucuu 921 asGfsaagAfuagaagcAfgCfcaggasas 1280 CUUCCUGGCUGCUUCUAUCUU 1639 1136160.1 cuL96 g CU AD- cscsuggcUfgCfUfUfcuaucuuc 922 asAfsgaaGfauagaagCfaGfccaggsas 1281 UUCCUGGCUGCUUCUAUCUUC 1640 1136161.1 uuL96 a UU AD- csusggcuGfcUfUfCfuaucuucu 923 asAfsagaAfgauagaaGfcAfgccagsgs 1282 UCCUGGCUGCUUCUAUCUUCU 1641 1136162.1 uuL96 a UU AD- usgsgcugCfuUfCfUfaucuucu 924 asAfsaagAfagauagaAfgCfagccasgs 1283 CCUGGCUGCUUCUAUCUUCUU 1642 1136163.1 uuuL96 g UG AD- gscsugcuUfcUfAfUfcuucuuu 925 asGfscaaAfgaagauaGfaAfgcagcscs 1284 UGGCUGCUUCUAUCUUCUUUG 1643 1136164.1 gcuL96 a CC AD- csusgcuuCfuAfUfCfuucuuug 926 asGfsgcaAfagaagauAfgAfagcagscs 1285 GGCUGCUUCUAUCUUCUUUGC 1644 1136165.1 ccuL96 c CA AD- usgscuucUfaUfCfUfucuuugcc 927 asUfsggdCa(Agn)agaagaUfaGfaag 1286 GCUGCUUCUAUCUUCUUUGCC 1645 1136166.1 auL96 casgsc AU AD- gscsuucuAfuCfUfUfcuuugcca 928 asAfsugdGc(Agn)aagaagAfuAfgaa 1287 CUGCUUCUAUCUUCUUUGCCA 1646 1136167.1 uuL96 gcsasg UC AD- csusucuaUfcUfUfCfuuugccau 929 asGfsaudGg(C2p)aaagaaGfaUfagaa 1288 UGCUUCUAUCUUCUUUGCCAU 1647 1136168.1 cuL96 gscsa CA AD- ususcuauCfuUfCfUfuugccauc 930 asUfsgadTg(G2p)caaagaAfgAfuag 1289 GCUUCUAUCUUCUUUGCCAUC 1648 1136169.1 auL96 aasgsc AA AD- uscsuaucUfuCfUfUfugccauca 931 asUfsugdAu(G2p)gcaaagAfaGfaua 1290 CUUCUAUCUUCUUUGCCAUCA 1649 1136170.1 auL96 gasasg AA AD- csusaucuUfcUfUfUfgccaucaa 932 asUfsuudGa(Tgn)ggcaaaGfaAfgau 1291 UUCUAUCUUCUUUGCCAUCAA 1650 1136171.1 auL96 agsasa AG AD- usasucuuCfuUfUfGfccaucaaa 933 asCfsuuuGfauggcaaAfgAfagauasgs 1292 UCUAUCUUCUUUGCCAUCAAA 1651 1136172.1 guL96 a GA AD- asuscuucUfuUfGfCfcaucaaag 934 asUfscudTu(G2p)auggcaAfaGfaag 1293 CUAUCUUCUUUGCCAUCAAAG 1652 1136173.1 auL96 ausasg AU AD- csusucuuUfgCfCfAfucaaagau 935 asCfsaucUfuugauggCfaAfagaagsas 1294 AUCUUCUUUGCCAUCAAAGAU 1653 1136174.1 guL96 u GC AD- gsasgcucAfgCfAfCfacagguaa 936 asAfsuudAc(C2p)ugugugCfuGfagc 1295 UCGAGCUCAGCACACAGGUAA 1654 1136175.1 uuL96 ucsgsa UA AD- csuscagcAfcAfCfAfgguaauaa 937 asGfsuuaUfuaccuguGfuGfcugagscs 1296 AGCUCAGCACACAGGUAAUAA 1655 1136176.1 cuL96 u CG AD- uscsagcaCfaCfAfGfguaauaac 938 asCfsguuAfuuaccugUfgUfgcugasgs 1297 GCUCAGCACACAGGUAAUAAC 1656 1136177.1 guL96 c GU AD- csasgcacAfcAfGfGfuaauaacg 939 asAfscguUfauuaccuGfuGfugcugsas 1298 CUCAGCACACAGGUAAUAACG 1657 1136178.1 uuL96 g UG AD- ascsagguAfaUfAfAfcgugaagg 940 asUfsccdTu(C2p)acguuaUfuAfccu 1299 ACACAGGUAAUAACGUGAAG 1658 1136179.1 auL96 gusgsu GAA AD- csasgguaAfuAfAfCfgugaagga 941 asUfsucdCu(Tgn)cacguuAfuUfacc 1300 CACAGGUAAUAACGUGAAGG 1659 1136180.1 auL96 ugsusg AAC AD- asgsguaaUfaAfCfGfugaaggaa 942 asGfsuudCc(Tgn)ucacguUfaUfuacc 1301 ACAGGUAAUAACGUGAAGGA 1660 1136181.1 cuL96 usgsu ACU AD- asusaacgUfgAfAfGfgaacucuu 943 asGfsaadGa(G2p)uuccuuCfaCfguu 1302 UAAUAACGUGAAGGAACUCU 1661 1136182.1 cuL96 aususa UCC AD- usasacguGfaAfGfGfaacucuuc 944 asGfsgadAg(Agn)guuccuUfcAfcgu 1303 AAUAACGUGAAGGAACUCUUC 1662 1136183.1 cuL96 uasusu CG AD- cscscagaAfaAfCfUfgcaaaccc 945 asAfsggdGu(Tgn)ugcaguUfuUfcug 1304 GUCCCAGAAAACUGCAAACCC 1663 1136184.1 uuL96 ggsasc UG AD- csascagaAfcAfUfGfgaucuauu 946 asUfsaadTa(G2p)auccauGfuUfcug 1305 ACCACAGAACAUGGAUCUAUU 1664 1136185.1 auL96 ugsgsu AA AD- ascsagaaCfaUfGfGfaucuauua 947 asUfsuaaUfagauccaUfgUfucugusgs 1306 CCACAGAACAUGGAUCUAUUA 1665 1136186.1 auL96 g AA AD- csasgaacAfuGfGfAfucuauuaa 948 asUfsuuaAfuagauccAfuGfuucugsus 1307 CACAGAACAUGGAUCUAUUAA 1666 1136187.1 auL96 g AG AD- asgsaacaUfgGfAfUfcuauuaaa 949 asCfsuuuAfauagaucCfaUfguucusgs 1308 ACAGAACAUGGAUCUAUUAA 1667 1136188.1 guL96 u AGU AD- gsasacauGfgAfUfCfuauuaaag 950 asAfscuuUfaauagauCfcAfuguucsus 1309 CAGAACAUGGAUCUAUUAAA 1668 1136189.1 uuL96 g GUC AD- asascaugGfaUfCfUfauuaaagu 951 asGfsacuUfuaauagaUfcCfauguuscs 1310 AGAACAUGGAUCUAUUAAAG 1669 1136190.1 cuL96 u UCA AD- ascsauggAfuCfUfAfuuaaaguc 952 asUfsgadCu(Tgn)uaauagAfuCfcau 1311 GAACAUGGAUCUAUUAAAGU 1670 1136191.1 auL96 gususc CAC AD- csasuggaUfcUfAfUfuaaaguca 953 asGfsugdAc(Tgn)uuaauaGfaUfcca 1312 AACAUGGAUCUAUUAAAGUC 1671 1136192.1 cuL96 ugsusu ACA AD- asusggauCfuAfUfUfaaagucac 954 asUfsgudGa(C2p)uuuaauAfgAfucc 1313 ACAUGGAUCUAUUAAAGUCAC 1672 1136193.1 auL96 ausgsu AG AD- usgsgaucUfaUfUfAfaagucaca 955 asCfsugdTg(Agn)cuuuaaUfaGfauc 1314 CAUGGAUCUAUUAAAGUCACA 1673 1136194.1 guL96 casusg GA AD- gsgsaucuAfuUfAfAfagucacag 956 asUfscudGu(G2p)acuuuaAfuAfgau 1315 AUGGAUCUAUUAAAGUCACA 1674 1136195.1 auL96 ccsasu GAA AD- gsasucuaUfuAfAfAfgucacaga 957 asUfsucdTg(Tgn)gacuuuAfaUfaga 1316 UGGAUCUAUUAAAGUCACAG 1675 1136196.1 auL96 ucscsa AAU AD- ascsaaugAfuAfAfGfcaaauuca 958 asUfsugaAfuuugcuuAfuCfauugusg 1317 ACACAAUGAUAAGCAAAUUCA 1676 1136197.1 auL96 su AA AD- csasaugaUfaAfGfCfaaauucaa 959 asUfsuudGa(Agn)uuugcuUfaUfcau 1318 CACAAUGAUAAGCAAAUUCAA 1677 1136198.1 auL96 ugsusg AA AD- asusgauaAfgCfAfAfauucaaaa 960 asGfsuudTu(G2p)aauuugCfuUfauc 1319 CAAUGAUAAGCAAAUUCAAA 1678 1136199.1 cuL96 aususg ACU AD- asusgccuAfaAfUfGfgugaauau 961 asCfsauaUfucaccauUfuAfggcausas 1320 UUAUGCCUAAAUGGUGAAUA 1679 1136200.1 guL96 a UGC AD- usgsccuaAfaUfGfGfugaauaug 962 asGfscauAfuucaccaUfuUfaggcasus 1321 UAUGCCUAAAUGGUGAAUAU 1680 1136201.1 cuL96 a GCA AD- gscscuaaAfuGfGfUfgaauaugc 963 asUfsgcdAu(Agn)uucaccAfuUfuag 1322 AUGCCUAAAUGGUGAAUAUG 1681 1136202.1 auL96 gcsasu CAA AD- cscsuaaaUfgGfUfGfaauaugca 964 asUfsugdCa(Tgn)auucacCfaUfuuag 1323 UGCCUAAAUGGUGAAUAUGC 1682 1136203.1 auL96 gscsa AAU AD- csusaaauGfgUfGfAfauaugcaa 965 asAfsuudGc(Agn)uauucaCfcAfuuu 1324 GCCUAAAUGGUGAAUAUGCA 1683 1136204.1 uuL96 agsgsc AUU AD- asasugguGfaAfUfAfugcaauua 966 asCfsuaaUfugcauauUfcAfccauusus 1325 UAAAUGGUGAAUAUGCAAUU 1684 1136205.1 guL96 a AGG AD- csgsggaaGfgGfUfUfugugcua 967 asAfsaudAg(C2p)acaaacCfcUfuccc 1326 GUCGGGAAGGGUUUGUGCUA 1685 1136206.1 uuuL96 gsasc UUC AD- gsgsgaagGfgUfUfUfgugcuau 968 asGfsaadTa(G2p)cacaaaCfcCfuucc 1327 UCGGGAAGGGUUUGUGCUAU 1686 1136207.1 ucuL96 csgsa UCC AD- gsgsaaggGfuUfUfGfugcuauu 969 asGfsgaaUfagcacaaAfcCfcuuccscs 1328 CGGGAAGGGUUUGUGCUAUU 1687 1136208.1 ccuL96 g CCC AD- gsusauaaCfcUfCfAfaguucuga 970 asAfsucaGfaacuugaGfgUfuauacsas 1329 CUGUAUAACCUCAAGUUCUGA 1688 1136209.1 uuL96 g UG AD- usasuaacCfuCfAfAfguucugau 971 asCfsaudCa(G2p)aacuugAfgGfuua 1330 UGUAUAACCUCAAGUUCUGAU 1689 1136210.1 guL96 uascsa GG AD- cscsucaaGfuUfCfUfgauggugu 972 asGfsacdAc(C2p)aucagaAfcUfuga 1331 AACCUCAAGUUCUGAUGGUGU 1690 1136211.1 cuL96 ggsusu CU AD- csasaguuCfuGfAfUfggugucu 973 asAfscadGa(C2p)accaucAfgAfacuu 1332 CUCAAGUUCUGAUGGUGUCUG 1691 1136212.1 guuL96 gsasg UC AD- cscsacaaAfcCfUfCfuagaagcu 974 asAfsagdCu(Tgn)cuagagGfuUfugu 1333 UCCCACAAACCUCUAGAAGCU 1692 1136213.1 uuL96 ggsgsa UA AD- ascsaaacCfuCfUfAfgaagcuua 975 asUfsuadAg(C2p)uucuagAfgGfuuu 1334 CCACAAACCUCUAGAAGCUUA 1693 1136214.1 auL96 gusgsg AA AD- asasaccuCfuAfGfAfagcuuaaa 976 asGfsuuuAfagcuucuAfgAfgguuusg 1335 ACAAACCUCUAGAAGCUUAAA 1694 1136215.1 cuL96 su CC AD- asasccucUfaGfAfAfgcuuaaac 977 asGfsguuUfaagcuucUfaGfagguusus 1336 CAAACCUCUAGAAGCUUAAAC 1695 1136216.1 cuL96 g CG AD- usgsgccuUfcAfAfAfccaaugaa 978 asGfsuucAfuugguuuGfaAfggccasg 1337 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asUfsgcdAg(Tgn)aaaaugGfaUfcaca 1427 UCCUGUGAUCCAUUUUACUGC 1786 1136307.1 auL96 gsgsa AA AD- csusauaaAfuAfUfAfugaccuga 1069 asUfsucdAg(G2p)ucauauAfuUfuau 1428 GCCUAUAAAUAUAUGACCUGA 1787 1136308.1 auL96 agsgsc AA AD- usgsaccuGfaAfAfAfcuccaguu 1070 asUfsaadCu(G2p)gaguuuUfcAfggu 1429 UAUGACCUGAAAACUCCAGUU 1788 1136309.1 auL96 casusa AC AD- gsasccugAfaAfAfCfuccaguua 1071 asGfsuadAc(Tgn)ggaguuUfuCfagg 1430 AUGACCUGAAAACUCCAGUUA 1789 1136310.1 cuL96 ucsasu CA AD- asasggauCfuGfCfAfgcuaucua 1072 asUfsuadGa(Tgn)agcugcAfgAfucc 1431 UAAAGGAUCUGCAGCUAUCUA 1790 1136311.1 auL96 uususa AG AD- asgsgaucUfgCfAfGfcuaucuaa 1073 asCfsuuaGfauagcugCfaGfauccusus 1432 AAAGGAUCUGCAGCUAUCUAA 1791 1136312.1 guL96 u GG AD- gscsuaucUfaAfGfGfcuugguu 1074 asAfsaaaCfcaagccuUfaGfauagcsus 1433 CAGCUAUCUAAGGCUUGGUUU 1792 1136313.1 uuuL96 g UC AD- csusaucuAfaGfGfCfuugguuu 1075 asGfsaaaAfccaagccUfuAfgauagscs 1434 AGCUAUCUAAGGCUUGGUUU 1793 1136314.1 ucuL96 u UCU AD- asuscuaaGfgCfUfUfgguuuuc 1076 asAfsagaAfaaccaagCfcUfuagausas 1435 CUAUCUAAGGCUUGGUUUUCU 1794 1136315.1 uuuL96 g UA AD- uscsuaagGfcUfUfGfguuuucu 1077 asUfsaadGa(Agn)aaccaaGfcCfuuag 1436 UAUCUAAGGCUUGGUUUUCU 1795 1136316.1 uauL96 asusa UAC AD- csusaaggCfuUfGfGfuuuucuu 1078 asGfsuaaGfaaaaccaAfgCfcuuagsas 1437 AUCUAAGGCUUGGUUUUCUU 1796 1136317.1 acuL96 u ACU AD- usasaggcUfuGfGfUfuuucuua 1079 asAfsguaAfgaaaaccAfaGfccuuasgs 1438 UCUAAGGCUUGGUUUUCUUAC 1797 1136318.1 cuuL96 a UG AD- gsgscuugGfuUfUfUfcuuacug 1080 asGfsacdAg(Tgn)aagaaaAfcCfaagc 1439 AAGGCUUGGUUUUCUUACUG 1798 1136319.1 ucuL96 csusu UCA AD- gscsuuggUfuUfUfCfuuacugu 1081 asUfsgadCa(G2p)uaagaaAfaCfcaag 1440 AGGCUUGGUUUUCUUACUGUC 1799 1136320.1 cauL96 cscsu AU AD- csusugguUfuUfCfUfuacuguc 1082 asAfsugdAc(Agn)guaagaAfaAfcca 1441 GGCUUGGUUUUCUUACUGUCA 1800 1136321.1 auuL96 agscsc UA AD- ususgguuUfuCfUfUfacuguca 1083 asUfsaudGa(C2p)aguaagAfaAfacca 1442 GCUUGGUUUUCUUACUGUCAU 1801 1136322.1 uauL96 asgsc AU AD- usgsguuuUfcUfUfAfcugucau 1084 asAfsuadTg(Agn)caguaaGfaAfaacc 1443 CUUGGUUUUCUUACUGUCAUA 1802 1136323.1 auuL96 asasg UG AD- gsgsuuuuCfuUfAfCfugucaua 1085 asCfsauaUfgacaguaAfgAfaaaccsas 1444 UUGGUUUUCUUACUGUCAUA 1803 1136324.1 uguL96 a UGA AD- ususuucuUfaCfUfGfucauauga 1086 asAfsucaUfaugacagUfaAfgaaaascs 1445 GGUUUUCUUACUGUCAUAUG 1804 1136325.1 uuL96 c AUA AD- ususucuuAfcUfGfUfcauauga 1087 asUfsaucAfuaugacaGfuAfagaaasas 1446 GUUUUCUUACUGUCAUAUGA 1805 1136326.1 uauL96 c UAC AD- uscsuuacUfgUfCfAfuaugauac 1088 asGfsgudAu(C2p)auaugaCfaGfuaa 1447 UUUCUUACUGUCAUAUGAUAC 1806 1136327.1 cuL96 gasasa CU AD- csusuacuGfuCfAfUfaugauacc 1089 asAfsgguAfucauaugAfcAfguaagsas 1448 UUCUUACUGUCAUAUGAUACC 1807 1136328.1 uuL96 a UG AD- uscsugcgGfuUfGfGfuaaagaga 1090 asUfsucdTc(Tgn)uuaccaAfcCfgcag 1449 UUUCUGCGGUUGGUAAAGAG 1808 1136329.1 auL96 asasa AAC AD- usgsgugaUfuUfAfAfucccuaca 1091 asAfsugdTa(G2p)ggauuaAfaUfcac 1450 AAUGGUGAUUUAAUCCCUACA 1809 1136330.1 uuL96 casusu UG AD- gsgsugauUfuAfAfUfcccuacau 1092 asCfsaugUfagggauuAfaAfucaccsas 1451 AUGGUGAUUUAAUCCCUACAU 1810 1136331.1 guL96 u GG AD- usgsuauaAfaUfCfCfaaccuucu 1093 asCfsagaAfgguuggaUfuUfauacasgs 1452 ACUGUAUAAAUCCAACCUUCU 1811 1136332.1 guL96 u GC AD- gsusgcuuGfaUfGfUfcacuaaua 1094 asUfsuadTu(Agn)gugacaUfcAfagc 1453 UGGUGCUUGAUGUCACUAAU 1812 1136333.1 auL96 acscsa AAA AD- usgscuugAfuGfUfCfacuaauaa 1095 asUfsuuaUfuagugacAfuCfaagcascs 1454 GGUGCUUGAUGUCACUAAUA 1813 1136334.1 auL96 c AAU AD- gscsuugaUfgUfCfAfcuaauaaa 1096 asAfsuuuAfuuagugaCfaUfcaagcsas 1455 GUGCUUGAUGUCACUAAUAA 1814 1136335.1 uuL96 c AUG AD- asusgucaCfuAfAfUfaaaugaaa 1097 asGfsuudTc(Agn)uuuauuAfgUfgac 1456 UGAUGUCACUAAUAAAUGAA 1815 1136336.1 cuL96 auscsa ACU AD- usgsucacUfaAfUfAfaaugaaac 1098 asAfsgudTu(C2p)auuuauUfaGfuga 1457 GAUGUCACUAAUAAAUGAAA 1816 1136337.1 uuL96 casusc CUG AD- usasauaaAfuGfAfAfacugucag 1099 asGfscudGa(C2p)aguuucAfuUfuau 1458 ACUAAUAAAUGAAACUGUCA 1817 1136338.1 cuL96 uasgsu GCU

TABLE 4 Xanthine Dehydrogenase dsRNA Agent In Vitro Single Dose Screens in Primary Human Hepatocytes PHH 500 nM 100 nM 10 nM % of Avg % of Avg % of Avg Message Message Message Duplex ID Remaining STDEV Remaining STDEV Remaining STDEV AD-1135979.1 78.6 8.5 81.4 6.5 68.6 25.5 AD-1135980.1 98.2 10.7 92.0 18.8 64.8 1.2 AD-1135981.1 79.5 14.5 92.4 19.2 60.7 9.1 AD-1135982.1 66.7 6.7 80.3 6.8 67.5 4.1 AD-1135983.1 68.7 9.7 75.0 3.5 62.8 6.8 AD-1135984.1 69.4 14.9 66.0 5.0 68.3 13.1 AD-1135985.1 59.8 10.9 69.5 26.8 69.2 9.4 AD-1135986.1 94.2 30.7 54.0 21.9 58.0 7.8 AD-1135987.1 29.0 5.3 47.3 10.4 44.4 7.8 AD-1135988.1 82.4 15.5 85.9 4.8 63.9 1.9 AD-1135989.1 36.6 8.6 58.0 10.7 52.9 3.2 AD-1135990.1 86.8 31.4 94.2 5.1 73.2 16.0 AD-1135991.1 36.0 6.7 45.8 9.5 54.3 9.2 AD-1135992.1 66.2 18.6 81.8 9.3 78.3 14.7 AD-1135993.1 68.7 16.2 56.9 17.0 62.7 7.4 AD-1135994.1 60.5 21.8 71.3 22.4 70.5 18.1 AD-1135995.1 51.2 5.0 76.5 6.1 54.8 6.4 AD-1135996.1 38.6 8.3 56.5 8.9 54.8 7.4 AD-1135997.1 63.7 13.1 78.9 6.3 67.4 7.1 AD-1135998.1 45.7 6.7 42.2 3.3 59.4 18.4 AD-1135999.1 39.8 6.5 61.4 10.2 67.1 14.9 AD-1136000.1 59.6 8.2 60.7 15.3 58.8 4.7 AD-1136001.1 29.4 7.6 42.2 11.7 47.3 3.1 AD-1136002.1 35.2 6.9 35.1 6.1 36.4 6.1 AD-1136003.1 59.4 9.1 55.3 8.4 51.2 7.4 AD-1136004.1 39.8 4.4 59.7 4.9 62.2 12.8 AD-1136005.1 74.9 7.7 97.7 35.8 81.3 11.8 AD-1136006.1 53.9 7.3 87.2 11.7 68.6 11.2 AD-1136007.1 34.0 7.7 55.2 5.3 55.5 2.9 AD-1136008.1 26.6 9.2 46.9 22.0 55.4 9.7 AD-1136009.1 41.9 11.4 69.5 25.5 65.4 10.1 AD-1136010.1 86.3 20.6 100.7 20.6 77.0 10.3 AD-1136011.1 84.9 11.0 91.8 14.5 63.6 6.0 AD-1136012.1 43.6 11.3 59.6 4.9 67.0 8.4 AD-1136013.1 74.1 8.8 96.5 30.1 68.6 13.1 AD-1136014.1 82.1 16.3 83.9 13.5 68.5 6.9 AD-1136015.1 47.4 6.1 78.1 3.4 67.1 10.2 AD-1136016.1 45.3 19.8 69.7 10.5 63.3 5.7 AD-1136017.1 41.3 5.8 77.8 14.2 59.2 4.0 AD-1136018.1 47.4 10.9 64.5 11.1 64.4 10.8 AD-1136019.1 85.6 9.7 97.4 9.3 67.8 2.9 AD-1136020.1 54.7 8.5 68.9 9.4 59.7 1.8 AD-1136021.1 40.9 16.6 60.7 5.1 71.3 24.0 AD-1136022.1 43.0 8.0 57.9 12.5 62.5 9.2 AD-1136023.1 46.1 20.5 71.9 8.1 60.6 4.8 AD-1136024.1 43.4 3.0 68.9 12.2 57.3 15.8 AD-1136025.1 61.1 9.2 74.0 6.2 69.6 13.1 AD-1136026.1 108.6 14.0 107.0 13.2 75.9 11.1 AD-1136027.1 120.4 32.7 104.1 23.0 77.3 11.7 AD-1136028.1 84.3 14.0 101.4 13.8 71.1 2.5 AD-1136029.1 120.4 32.1 94.0 7.1 86.3 17.7 AD-1136030.1 37.1 4.1 48.1 4.1 56.4 4.2 AD-1136031.1 55.4 26.1 75.1 14.6 69.7 16.4 AD-1136032.1 33.4 7.4 47.8 10.1 38.4 3.3 AD-1136033.1 57.6 20.1 77.1 7.3 60.6 4.2 AD-1136034.1 51.7 9.7 59.4 9.0 63.8 6.7 AD-1136035.1 58.2 10.8 89.8 10.8 64.4 3.0 AD-1136036.1 50.8 6.1 63.8 4.7 65.1 12.6 AD-1136037.1 41.8 7.7 50.4 3.1 64.3 17.4 AD-1136038.1 29.8 12.3 46.9 2.6 46.6 8.7 AD-1136039.1 28.7 11.8 37.0 5.6 64.7 29.0 AD-1136040.1 49.1 7.4 87.9 12.5 57.7 21.1 AD-1136041.1 66.6 18.3 99.9 8.1 78.2 14.9 AD-1136042.1 45.1 15.3 72.3 5.2 77.1 17.8 AD-1136043.1 37.3 21.7 53.9 6.4 60.2 9.1 AD-1136044.1 74.8 24.4 106.8 8.2 76.4 8.0 AD-1136045.1 43.6 10.2 92.3 15.6 69.8 12.7 AD-1136046.1 36.5 10.3 66.8 5.5 75.4 12.4 AD-1136047.1 40.5 10.3 71.0 9.0 67.4 19.4 AD-1136048.1 48.8 14.4 60.1 7.6 57.8 7.1 AD-1136049.1 82.5 19.0 91.0 17.0 63.6 6.6 AD-1136050.1 50.0 4.6 73.8 14.9 62.4 4.0 AD-1136051.1 28.7 1.3 44.3 7.6 47.7 8.2 AD-1136052.1 27.2 7.6 55.7 11.4 49.6 3.7 AD-1136053.1 33.6 22.8 49.1 7.9 54.4 4.7 AD-1136054.1 41.4 21.2 61.3 6.3 55.8 6.9 AD-1136055.1 58.3 16.1 78.4 6.8 63.1 9.9 AD-1136056.1 50.5 13.4 76.7 10.5 65.5 11.3 AD-1136057.1 47.0 13.0 70.4 15.6 55.4 2.8 AD-1136058.1 47.9 10.2 83.8 6.9 65.6 11.6 AD-1136059.1 76.2 14.1 95.4 17.7 67.3 11.0 AD-1136060.1 48.0 17.1 79.8 14.6 68.9 14.5 AD-1136061.1 28.0 5.2 53.9 10.6 53.8 13.6 AD-1136062.1 42.1 10.4 55.4 7.3 56.7 4.8 AD-1136063.1 90.1 40.7 79.3 2.8 62.9 11.6 AD-1136064.1 58.7 14.6 77.9 3.2 72.4 16.2 AD-1136065.1 78.2 16.0 83.8 11.7 62.9 6.1 AD-1136066.1 50.9 17.0 74.0 7.0 64.3 13.4 AD-1136067.1 60.6 11.1 78.6 5.1 60.6 7.6 AD-1136068.1 46.9 20.3 64.2 8.0 57.4 3.4 AD-1136069.1 33.5 10.4 34.8 10.9 54.5 4.7 AD-1136070.1 33.7 5.6 38.2 6.2 55.9 12.2 AD-1136071.1 43.7 7.9 47.8 5.6 82.7 10.9 AD-1136072.1 53.1 16.0 52.0 15.3 82.5 4.1 AD-1136073.1 37.8 9.0 38.5 9.0 56.7 11.1 AD-1136074.1 59.0 7.7 56.7 13.9 53.4 14.8 AD-1136075.1 25.9 5.2 36.6 6.8 32.5 10.8 AD-1136076.1 38.0 11.5 40.0 15.3 42.6 9.8 AD-1136077.1 33.2 4.7 39.2 10.9 70.9 15.7 AD-1136078.1 53.5 7.7 59.0 4.6 84.5 5.9 AD-1136079.1 52.0 18.0 63.3 13.1 91.1 12.3 AD-1136080.1 49.3 19.0 67.8 16.7 97.7 18.9 AD-1136081.1 29.0 5.4 40.0 13.8 63.7 12.3 AD-1136082.1 28.9 19.2 26.2 4.5 39.4 5.4 AD-1136083.1 17.6 1.9 18.7 2.7 24.6 2.9 AD-1136084.1 27.4 3.7 32.3 7.8 43.4 8.6 AD-1136085.1 35.0 8.9 41.8 18.1 83.9 20.0 AD-1136086.1 74.4 23.0 78.3 11.0 102.6 14.3 AD-1136087.1 67.6 29.6 33.7 7.6 81.8 16.9 AD-1136088.1 27.4 4.9 52.3 2.4 78.4 5.9 AD-1136089.1 37.2 18.0 33.6 1.7 61.0 15.7 AD-1136090.1 56.0 24.2 67.8 5.6 77.0 5.9 AD-1136091.1 23.2 4.3 32.6 13.8 44.2 7.7 AD-1136092.1 35.3 11.0 58.7 23.6 87.8 22.4 AD-1136093.1 44.8 11.5 43.7 14.0 81.5 18.1 AD-1136094.1 47.6 12.0 47.8 9.7 94.1 28.6 AD-1136095.1 69.2 14.9 69.7 8.4 96.6 11.1 AD-1136096.1 67.0 21.6 73.8 5.2 94.4 4.7 AD-1136097.1 84.8 9.5 76.0 8.6 93.5 15.0 AD-1136098.1 58.1 3.2 49.0 3.3 74.4 15.8 AD-1136099.1 63.7 17.1 72.7 21.6 112.8 14.8 AD-1136100.1 40.7 4.6 39.7 9.2 76.0 13.5 AD-1136101.1 49.5 11.9 39.8 3.4 67.2 2.7 AD-1136102.1 49.3 3.4 40.5 4.4 77.7 11.2 AD-1136103.1 44.7 6.8 49.9 4.6 82.0 11.3 AD-1136104.1 56.9 13.7 52.9 5.4 87.6 5.3 AD-1136105.1 69.7 29.4 60.4 10.7 79.9 9.4 AD-1136106.1 28.3 8.4 36.9 7.8 54.4 9.8 AD-1136107.1 35.4 8.3 61.2 18.1 84.4 3.2 AD-1136108.1 49.1 20.7 49.7 12.8 75.4 17.4 AD-1136109.1 65.0 17.5 63.6 10.2 95.5 10.9 AD-1136110.1 121.2 18.0 91.7 14.5 102.9 7.7 AD-1136111.1 85.6 9.1 80.8 17.2 96.8 12.5 AD-1136112.1 49.6 3.0 46.6 8.9 72.9 12.9 AD-1136114.1 45.2 9.2 37.7 18.3 71.0 4.6 AD-1136115.1 76.2 8.7 80.3 16.2 94.8 6.0 AD-1136116.1 108.7 15.0 105.2 23.4 119.1 31.1 AD-1136117.1 45.8 8.8 37.1 4.1 56.7 6.1 AD-1136118.1 51.8 12.1 43.7 10.2 64.7 8.0 AD-1136119.1 68.7 9.8 53.1 7.7 83.8 6.9 AD-1136120.1 54.9 7.9 45.6 5.9 80.8 20.4 AD-1136121.1 52.4 19.3 64.3 12.8 79.1 19.4 AD-1136122.1 103.8 15.0 91.1 13.4 113.7 22.7 AD-1136123.1 57.7 5.0 62.6 13.3 95.9 7.3 AD-1136124.1 69.7 5.7 74.6 5.3 96.5 21.5 AD-1136125.1 47.0 9.3 49.2 5.0 75.2 20.0 AD-1136126.1 58.9 1.7 57.8 13.0 84.7 9.7 AD-1136127.1 50.2 11.1 33.2 5.1 58.2 5.2 AD-1136128.1 111.8 17.4 81.9 3.7 104.2 25.1 AD-1136129.1 77.6 34.6 100.1 45.8 79.2 12.9 AD-1136130.1 51.5 2.4 62.2 9.0 98.9 17.0 AD-1136131.1 78.0 13.8 66.1 4.7 95.2 12.6 AD-1136132.1 73.5 9.3 68.8 3.1 104.0 12.2 AD-1136133.1 65.9 4.8 66.8 16.8 89.7 9.7 AD-1136134.1 72.8 13.6 57.5 4.6 84.9 5.0 AD-1136135.1 57.0 17.8 59.3 9.0 86.4 6.9 AD-1136136.1 49.1 25.8 58.5 4.6 63.5 7.5 AD-1136137.1 50.4 8.5 57.1 18.7 91.4 19.0 AD-1136138.1 83.8 21.2 72.2 13.5 92.9 7.0 AD-1136139.1 44.6 9.9 39.8 7.5 60.0 10.4 AD-1136140.1 75.1 8.6 64.3 2.4 89.1 11.8 AD-1136141.1 36.6 3.5 30.3 2.9 61.7 13.8 AD-1136142.1 38.7 11.0 38.3 7.1 63.9 4.8 AD-1136143.1 34.9 13.9 46.9 16.2 66.0 23.0 AD-1136144.1 39.1 6.9 50.6 2.9 75.2 16.3 AD-1136145.1 58.8 3.7 55.3 7.0 84.5 19.3 AD-1136146.1 69.4 4.6 67.7 12.3 93.8 12.6 AD-1136147.1 118.8 30.5 93.9 15.8 99.0 8.6 AD-1136148.1 65.9 17.7 62.0 4.9 82.9 3.2 AD-1136149.1 60.1 23.6 43.5 4.7 81.8 7.4 AD-1136150.1 30.2 6.2 43.5 12.6 86.1 10.5 AD-1136151.1 47.3 10.5 70.6 23.5 66.4 9.4 AD-1136152.1 56.8 25.0 63.5 18.1 70.5 25.6 AD-1136153.1 52.3 8.3 49.4 13.5 77.7 7.8 AD-1136154.1 47.3 17.6 49.5 5.8 85.6 20.7 AD-1136155.1 106.6 1.5 76.3 15.7 92.6 6.2 AD-1136156.1 36.6 9.6 50.8 32.7 65.3 8.2 AD-1136157.1 30.2 16.8 36.8 15.8 67.0 8.9 AD-1136158.1 54.9 10.5 68.4 15.9 78.3 18.4 AD-1136159.1 27.6 5.5 50.6 19.2 93.9 20.2 AD-1136160.1 31.7 5.8 53.7 16.2 121.0 34.1 AD-1136161.1 32.0 5.4 62.0 25.2 68.9 9.8 AD-1136162.1 27.5 4.8 46.0 10.9 98.4 37.9 AD-1136163.1 21.6 7.4 33.8 6.6 52.8 26.7 AD-1136164.1 47.9 25.0 64.4 13.8 81.3 9.8 AD-1136165.1 31.9 6.9 62.1 2.0 56.8 11.0 AD-1136166.1 20.5 3.9 35.5 6.5 52.0 26.5 AD-1136167.1 55.5 10.9 88.1 31.7 87.4 8.2 AD-1136168.1 53.0 3.7 64.5 18.7 91.7 31.7 AD-1136169.1 27.7 2.4 32.0 6.6 50.6 7.6 AD-1136170.1 28.0 2.5 39.1 16.3 61.7 6.3 AD-1136171.1 78.1 6.6 54.6 21.1 96.7 4.1 AD-1136172.1 43.7 9.8 58.0 19.0 82.1 7.5 AD-1136173.1 34.8 6.7 62.4 10.9 78.4 9.5 AD-1136174.1 29.3 6.9 59.8 15.4 61.3 22.5 AD-1136175.1 39.0 6.3 57.8 26.0 70.6 16.2 AD-1136176.1 71.2 7.3 64.8 22.4 107.7 18.8 AD-1136177.1 66.4 5.4 64.9 27.3 90.1 8.1 AD-1136178.1 47.5 8.5 61.3 17.3 87.4 8.7 AD-1136179.1 45.9 2.8 45.9 15.6 80.8 17.7 AD-1136180.1 56.6 18.0 59.3 7.1 66.9 5.1 AD-1136181.1 28.8 5.9 41.2 4.2 35.8 3.4 AD-1136182.1 66.9 34.9 82.1 28.4 129.7 48.8 AD-1136183.1 100.7 16.6 54.8 18.7 105.8 8.5 AD-1136184.1 72.6 9.2 51.5 8.0 113.2 16.5 AD-1136185.1 35.6 8.3 36.4 11.7 63.9 12.1 AD-1136186.1 40.0 14.4 35.5 13.1 49.2 8.6 AD-1136187.1 28.6 2.8 29.7 11.3 34.5 9.3 AD-1136188.1 42.9 6.3 53.5 11.6 74.4 49.5 AD-1136189.1 42.1 7.5 27.5 6.4 82.1 4.1 AD-1136190.1 52.0 21.2 51.7 20.4 102.1 6.2 AD-1136191.1 34.8 5.6 42.0 17.6 51.1 10.0 AD-1136192.1 53.3 7.0 47.9 12.6 62.1 3.1 AD-1136193.1 44.7 6.5 46.9 5.6 44.0 8.3 AD-1136194.1 41.4 5.9 58.6 17.5 60.4 16.8 AD-1136195.1 55.5 6.3 47.9 19.4 58.2 14.3 AD-1136196.1 37.3 4.0 45.8 11.9 47.8 11.2 AD-1136197.1 39.2 7.4 40.0 12.2 71.4 25.0 AD-1136198.1 68.5 16.0 78.5 25.7 91.1 11.8 AD-1136199.1 55.3 5.4 93.1 18.1 71.0 21.0 AD-1136200.1 70.6 4.0 89.1 9.9 75.5 23.2 AD-1136201.1 57.1 10.4 76.0 12.2 88.0 13.0 AD-1136202.1 69.9 15.3 68.6 6.4 53.2 11.9 AD-1136203.1 57.4 8.3 103.7 29.4 44.9 4.9 AD-1136204.1 38.2 7.2 55.3 8.9 104.7 41.6 AD-1136205.1 47.9 5.5 88.6 26.9 111.5 23.5 AD-1136206.1 81.7 13.0 109.4 16.9 107.2 13.7 AD-1136207.1 68.2 7.2 101.3 10.3 78.6 14.3 AD-1136208.1 71.8 4.9 102.4 18.6 76.5 33.1 AD-1136209.1 74.7 10.1 100.9 37.2 65.8 17.0 AD-1136210.1 56.3 2.9 73.5 19.8 57.7 10.7 AD-1136211.1 65.2 7.3 100.2 28.2 56.4 4.2 AD-1136212.1 68.0 9.0 80.4 27.1 95.4 30.6 AD-1136213.1 61.3 11.2 83.6 7.5 87.1 11.6 AD-1136214.1 42.3 7.1 64.9 10.9 67.7 12.0 AD-1136215.1 52.8 3.6 65.0 6.1 55.1 16.4 AD-1136216.1 69.3 8.4 97.1 12.4 77.4 15.7 AD-1136217.1 53.8 6.9 87.4 22.9 81.9 20.1 AD-1136218.1 54.8 7.2 71.0 21.4 66.1 9.4 AD-1136219.1 46.9 10.2 67.7 26.7 44.6 4.5 AD-1136220.1 53.5 18.0 53.3 17.1 71.6 48.4 AD-1136221.1 33.1 2.5 61.5 2.1 82.6 28.7 AD-1136222.1 79.1 6.2 88.6 8.3 65.8 16.5 AD-1136223.1 47.6 1.8 68.0 6.1 58.6 12.7 AD-1136224.1 40.2 4.4 63.3 7.5 50.3 16.6 AD-1136225.1 29.8 4.6 53.7 10.7 61.3 44.7 AD-1136226.1 46.1 5.2 68.0 23.6 63.3 23.9 AD-1136227.1 40.9 3.9 63.0 10.2 73.8 19.3 AD-1136228.1 51.9 4.9 96.7 7.6 87.7 22.9 AD-1136229.1 44.5 4.0 74.2 3.7 72.2 22.1 AD-1136230.1 47.2 10.2 59.5 3.9 61.6 14.2 AD-1136231.1 41.2 4.9 61.0 13.5 59.6 20.1 AD-1136232.1 38.2 3.0 54.9 10.1 47.9 7.5 AD-1136233.1 37.2 3.9 63.7 12.1 52.9 12.6 AD-1136234.1 41.4 6.6 48.2 6.9 81.9 30.6 AD-1136235.1 87.4 19.0 80.6 22.0 119.7 40.0 AD-1136236.1 64.4 12.3 81.2 7.2 85.9 35.2 AD-1136237.1 80.5 4.3 125.8 18.4 97.4 21.2 AD-1136238.1 72.1 9.4 85.2 13.8 136.5 56.4 AD-1136239.1 63.0 18.5 96.0 28.1 103.5 37.2 AD-1136240.1 45.1 5.4 70.7 10.4 54.5 15.1 AD-1136241.1 56.8 3.7 72.3 32.0 56.3 14.8 AD-1136242.1 40.6 11.9 63.1 28.3 88.7 39.4 AD-1136243.1 30.8 4.4 51.7 10.8 65.4 29.0 AD-1136244.1 63.3 2.2 90.1 36.7 95.2 36.8 AD-1136245.1 94.6 13.8 91.9 26.1 116.1 27.6 AD-1136246.1 70.0 3.9 76.0 20.1 73.2 13.3 AD-1136247.1 45.3 7.2 46.7 16.3 70.7 51.5 AD-1136248.1 63.1 4.0 73.3 19.8 57.4 23.2 AD-1136249.1 59.2 14.4 59.4 6.1 81.5 11.1 AD-1136250.1 64.4 13.2 67.4 9.5 95.8 4.8 AD-1136251.1 87.5 17.7 63.5 11.6 97.1 17.0 AD-1136252.1 60.1 7.2 63.5 7.9 88.6 27.3 AD-1136253.1 75.8 10.0 84.2 13.3 95.7 37.6 AD-1136254.1 64.6 11.0 59.6 7.9 91.7 9.5 AD-1136255.1 56.8 7.8 70.0 22.1 97.7 19.5 AD-1136256.1 24.7 2.9 38.0 4.3 88.3 24.6 AD-1136257.1 54.9 10.8 46.2 6.8 68.8 7.2 AD-1136258.1 71.1 4.1 64.2 8.4 95.1 11.8 AD-1136259.1 85.2 13.4 69.2 3.5 106.0 23.0 AD-1136260.1 59.0 9.7 60.9 7.5 95.2 21.8 AD-1136261.1 70.1 19.7 63.3 1.9 122.1 31.3 AD-1136262.1 68.1 9.8 67.6 4.5 105.6 11.9 AD-1136263.1 39.0 9.5 46.6 8.0 97.1 20.0 AD-1136264.1 26.3 2.8 37.6 9.3 72.9 19.0 AD-1136265.1 53.2 6.5 50.2 7.8 77.0 8.4 AD-1136266.1 79.7 14.9 65.4 9.1 99.3 7.4 AD-1136267.1 100.0 13.4 81.8 4.3 98.6 9.9 AD-1136268.1 54.4 4.0 55.7 7.5 91.8 23.7 AD-1136269.1 32.0 8.1 40.6 8.0 90.8 20.1 AD-1136270.1 37.5 10.0 50.7 16.5 81.7 19.1 AD-1136271.1 32.3 8.5 42.5 8.3 60.6 11.8 AD-1136272.1 57.7 6.4 67.2 9.9 78.6 6.4 AD-1136273.1 87.8 21.5 95.4 16.5 103.5 17.7 AD-1136274.1 80.1 9.4 69.6 7.3 119.3 16.5 AD-1136275.1 71.4 12.2 76.8 14.1 139.8 50.5 AD-1136276.1 61.3 18.8 63.9 11.2 91.0 15.3 AD-1136277.1 41.7 2.0 43.6 10.3 92.3 11.5 AD-1136278.1 30.2 5.0 45.4 5.2 77.8 6.3 AD-1136279.1 52.3 0.2 49.2 9.6 80.5 12.8 AD-1136280.1 65.8 6.6 62.5 5.6 90.5 16.8 AD-1136281.1 70.3 4.6 68.3 3.8 124.4 52.7 AD-1136282.1 64.0 9.5 55.3 5.9 94.2 2.1 AD-1136283.1 58.9 14.2 62.4 6.3 103.8 7.2 AD-1136284.1 53.3 7.0 79.0 14.2 148.3 31.4 AD-1136285.1 48.7 4.5 53.6 7.0 93.2 15.7 AD-1136286.1 29.4 1.9 50.9 11.8 58.6 6.9 AD-1136287.1 57.6 14.7 52.6 6.2 63.6 9.9 AD-1136288.1 96.4 35.9 74.5 7.6 98.4 13.6 AD-1136289.1 66.2 10.5 69.8 12.1 110.1 12.8 AD-1136290.1 59.7 19.2 54.8 3.9 108.6 10.5 AD-1136291.1 57.5 11.2 56.6 8.5 85.8 5.4 AD-1136292.1 57.3 6.2 59.8 6.7 109.5 9.9 AD-1136293.1 66.8 6.3 55.7 7.6 90.5 24.6 AD-1136294.1 86.8 11.5 68.2 4.1 103.1 15.7 AD-1136295.1 83.1 6.9 84.9 6.3 103.9 18.6 AD-1136296.1 90.8 8.5 67.7 5.9 124.8 20.9 AD-1136297.1 86.0 13.8 91.8 16.6 132.2 29.3 AD-1136298.1 116.6 18.2 73.3 8.6 140.8 57.1 AD-1136299.1 84.6 12.3 70.7 14.7 100.7 27.4 AD-1136300.1 78.1 12.2 73.0 17.7 111.8 27.6 AD-1136301.1 58.9 3.1 64.4 7.4 76.3 6.0 AD-1136302.1 72.2 12.6 76.2 16.9 111.1 24.3 AD-1136303.1 91.1 11.6 99.4 17.6 130.4 26.5 AD-1136304.1 113.3 17.8 90.2 16.8 129.1 22.4 AD-1136305.1 129.1 31.2 98.9 7.3 132.2 19.6 AD-1136306.1 114.5 12.4 91.2 5.5 115.6 8.2 AD-1136307.1 102.8 22.0 91.1 9.2 132.4 22.8 AD-1136308.1 53.3 7.5 55.0 7.1 65.8 12.9 AD-1136309.1 31.8 3.9 51.1 4.8 55.0 13.3 AD-1136310.1 77.5 10.6 55.6 8.8 79.3 14.8 AD-1136311.1 79.4 13.0 73.2 4.2 95.4 19.9 AD-1136312.1 93.1 9.0 75.6 15.4 102.9 20.8 AD-1136313.1 63.2 5.1 53.8 4.0 103.8 33.1 AD-1136314.1 62.9 7.5 54.4 7.4 67.1 13.3 AD-1136315.1 60.1 7.5 59.3 10.2 90.7 46.5 AD-1136316.1 39.6 8.1 56.6 9.3 81.4 8.6 AD-1136317.1 77.6 28.1 55.9 9.6 61.6 10.0 AD-1136318.1 73.8 12.7 58.3 4.1 98.9 37.1 AD-1136319.1 76.7 7.6 61.5 2.8 112.7 15.4 AD-1136320.1 80.1 23.9 61.2 6.0 109.4 18.5 AD-1136321.1 56.6 3.5 45.1 10.5 72.2 8.3 AD-1136322.1 63.0 11.0 44.2 11.9 71.4 9.7 AD-1136323.1 55.4 6.9 58.5 9.1 82.9 33.3 AD-1136324.1 53.8 12.8 55.5 17.5 59.5 18.1 AD-1136325.1 71.9 2.9 70.4 9.6 70.8 14.0 AD-1136326.1 64.3 5.6 59.0 11.8 63.3 18.4 AD-1136327.1 124.1 12.1 78.0 8.7 82.4 18.6 AD-1136328.1 68.2 15.9 57.0 7.0 74.3 2.4 AD-1136329.1 63.1 1.4 42.8 4.8 65.1 17.1 AD-1136330.1 81.2 11.6 58.3 15.0 59.9 8.6 AD-1136331.1 67.7 12.3 56.1 17.5 48.3 2.3 AD-1136332.1 77.2 11.5 61.1 8.7 60.2 43.9 AD-1136333.1 67.6 8.3 46.4 9.6 63.5 13.5 AD-1136334.1 54.7 4.7 47.0 6.7 49.5 19.6 AD-1136335.1 70.7 23.3 55.9 7.3 72.2 6.0 AD-1136336.1 61.4 16.0 47.3 9.7 48.6 3.0 AD-1136337.1 68.7 11.8 58.5 16.7 57.7 8.7 AD-1136338.1 78.3 18.3 57.4 13.1 64.1 15.3

TABLE 5 Xanthine Dehydrogenase dsRNA Agent In Vitro Single Dose Screens in Primary Cynomolgus Hepatocytes PCH 500 nM 100 nM 10 nM 0.1 nM % of Avg % of Avg % of Avg % of Avg Message Message Message Message Duplex ID Remaining STDEV Remaining STDEV Remaining STDEV Remaining STDEV AD- 138.0 20.2 110.0 11.0 94.0 82.2 113.3 10.0 1135979.1 AD- 60.3 11.1 124.1 15.0 152.1 57.1 124.6 23.3 1135980.1 AD- 115.1 33.6 112.6 19.9 173.7 5.1 132.5 6.6 1135981.1 AD- 100.6 20.8 140.8 25.5 119.8 74.8 146.2 17.9 1135982.1 AD- 103.9 29.8 138.0 20.5 181.2 36.9 116.5 27.6 1135983.1 AD- 104.4 31.9 138.2 33.1 93.9 74.7 105.6 4.1 1135984.1 AD- 72.3 0.4 149.0 11.5 131.7 32.2 92.2 9.0 1135985.1 AD- 101.6 34.7 122.8 34.9 160.1 42.9 107.8 2.1 1135986.1 AD- 77.8 7.0 99.5 37.7 102.4 24.9 96.8 1.4 1135987.1 AD- 107.4 22.1 141.2 40.1 118.1 22.1 103.6 7.9 1135988.1 AD- 76.6 28.0 154.9 44.7 134.2 15.6 117.2 30.5 1135989.1 AD- 110.7 26.7 115.4 32.1 172.5 27.7 118.0 18.5 1135990.1 AD- 89.0 18.5 117.4 40.9 118.0 37.5 109.0 28.3 1135991.1 AD- 82.2 22.3 132.5 15.0 131.4 17.8 113.9 8.5 1135992.1 AD- 84.9 17.3 137.0 22.9 107.0 18.4 112.8 6.2 1135993.1 AD- 94.1 3.2 101.9 23.4 124.7 25.3 109.1 24.7 1135994.1 AD- 93.4 21.3 111.5 28.2 120.0 14.0 87.7 12.5 1135995.1 AD- 85.0 6.0 157.7 64.6 142.4 17.5 134.1 30.0 1135996.1 AD- 67.4 6.9 117.1 35.1 128.1 33.4 125.0 20.4 1135997.1 AD- 77.2 11.3 131.9 62.5 99.5 27.0 111.0 19.9 1135998.1 AD- 75.6 11.2 94.3 4.3 102.1 20.1 103.1 23.0 1135999.1 AD- 82.0 11.1 125.5 37.5 106.1 13.5 118.5 14.2 1136000.1 AD- 59.7 5.8 103.7 32.6 94.7 38.1 110.8 2.2 1136001.1 AD- 60.2 4.1 92.6 20.9 127.4 14.7 96.5 14.6 1136002.1 AD- 66.1 8.7 102.6 31.8 116.8 15.5 124.7 18.1 1136003.1 AD- 77.0 18.8 85.8 32.3 134.9 22.7 140.4 22.5 1136004.1 AD- 88.5 10.2 86.9 41.1 113.1 28.5 101.3 29.5 1136005.1 AD- 83.2 2.1 99.4 40.8 89.2 11.1 95.8 7.1 1136006.1 AD- 56.8 16.1 116.8 44.8 121.3 23.8 103.6 13.2 1136007.1 AD- 79.6 17.1 93.4 27.4 155.3 32.6 81.5 25.5 1136008.1 AD- 65.8 4.8 95.4 34.1 156.8 25.5 101.6 15.2 1136009.1 AD- 87.1 18.6 81.0 24.9 94.1 34.9 109.8 12.7 1136010.1 AD- 82.3 5.7 93.9 40.1 94.0 19.6 130.1 19.9 1136011.1 AD- 71.5 14.2 114.2 12.5 116.8 38.3 124.7 9.9 1136012.1 AD- 68.9 8.8 129.5 46.1 91.6 6.2 119.2 9.8 1136013.1 AD- 73.4 10.5 68.6 10.6 106.6 56.2 104.1 7.0 1136014.1 AD- 71.8 4.9 90.5 16.0 98.2 41.2 101.7 16.1 1136015.1 AD- 78.6 5.7 82.7 10.9 130.1 140.2 120.4 4.7 1136016.1 AD- 59.2 6.3 83.5 24.7 82.3 56.2 81.9 1.2 1136017.1 AD- 57.7 12.0 64.2 19.9 105.4 11.3 98.8 22.8 1136018.1 AD- 67.2 10.5 93.1 25.7 102.7 15.4 107.9 10.1 1136019.1 AD- 75.4 19.4 87.3 38.5 101.6 22.5 126.2 13.2 1136020.1 AD- 61.3 3.6 65.8 6.1 77.1 14.4 113.4 17.8 1136021.1 AD- 59.2 22.6 75.0 13.3 116.4 48.4 107.3 17.4 1136022.1 AD- 71.9 21.4 76.1 14.2 100.8 38.3 91.6 16.3 1136023.1 AD- 82.1 8.1 81.4 35.1 116.6 5.3 83.7 4.5 1136024.1 AD- 75.8 30.1 107.4 32.7 115.5 5.3 87.4 15.6 1136025.1 AD- 69.2 11.6 127.8 46.6 97.5 24.8 95.5 18.1 1136026.1 AD- 86.9 8.3 118.5 19.4 124.4 28.7 116.4 24.3 1136027.1 AD- 84.2 30.1 139.7 31.6 95.7 7.5 125.4 18.6 1136028.1 AD- 75.1 8.4 82.5 19.0 74.3 8.3 93.3 12.2 1136029.1 AD- 44.5 5.6 84.2 33.7 120.3 51.2 99.0 14.8 1136030.1 AD- 66.2 9.8 96.0 46.4 117.0 67.0 80.8 16.5 1136031.1 AD- 62.1 16.0 65.9 13.8 91.3 60.9 82.6 11.3 1136032.1 AD- 61.5 7.8 83.1 16.5 123.6 23.4 102.5 17.5 1136033.1 AD- 54.3 16.6 80.6 13.2 101.9 7.7 105.9 6.3 1136034.1 AD- 78.6 19.3 93.1 20.7 112.6 15.2 117.2 4.8 1136035.1 AD- 79.4 5.2 77.1 19.8 159.8 86.1 105.2 23.1 1136036.1 AD- 59.5 16.2 89.9 32.7 71.5 11.2 100.6 3.3 1136037.1 AD- 56.9 11.1 68.5 10.1 98.6 41.5 95.9 15.9 1136038.1 AD- 49.4 7.2 61.8 25.1 153.5 77.6 74.7 13.8 1136039.1 AD- 52.1 13.1 84.6 35.1 155.3 9.7 72.5 7.5 1136040.1 AD- 71.9 17.8 75.0 29.6 89.4 10.9 91.1 9.1 1136041.1 AD- 85.2 25.0 76.0 6.6 120.5 23.3 113.6 14.8 1136042.1 AD- 89.3 21.7 89.1 45.3 110.0 4.8 92.9 23.7 1136043.1 AD- 81.6 16.4 66.5 15.2 101.0 38.9 92.7 23.4 1136044.1 AD- 68.2 3.6 75.1 13.4 84.4 22.8 92.8 12.6 1136045.1 AD- 75.8 7.4 90.3 25.3 71.8 12.2 71.5 3.7 1136046.1 AD- 94.8 5.1 98.7 40.9 134.8 25.3 70.6 2.4 1136047.1 AD- 78.6 18.3 84.4 22.1 92.4 15.9 71.7 4.1 1136048.1 AD- 91.4 32.0 84.3 19.5 111.3 17.6 113.8 13.0 1136049.1 AD- 64.1 15.1 89.4 27.9 101.3 45.3 97.5 23.8 1136050.1 AD- 44.3 7.4 67.5 28.3 86.1 18.7 107.4 25.9 1136051.1 AD- 62.3 17.5 69.8 32.2 86.2 19.2 77.4 21.0 1136052.1 AD- 57.0 5.6 77.8 20.7 131.4 35.3 68.9 22.1 1136053.1 AD- 59.2 17.7 75.6 21.8 97.2 26.8 80.9 6.7 1136054.1 AD- 74.7 13.5 94.8 20.5 74.2 19.1 88.6 11.4 1136055.1 AD- 70.8 19.5 85.1 20.8 117.7 26.0 92.1 4.1 1136056.1 AD- 78.2 3.3 125.3 24.0 127.3 31.9 93.4 12.6 1136057.1 AD- 73.7 15.3 64.5 13.5 113.2 21.3 81.7 23.1 1136058.1 AD- 84.5 9.5 90.3 28.2 118.2 61.6 81.6 15.8 1136059.1 AD- 72.5 14.4 70.5 22.8 80.7 13.0 65.1 14.1 1136060.1 AD- 60.8 20.7 84.8 15.8 99.7 27.0 54.9 1.6 1136061.1 AD- 96.7 25.3 60.3 2.9 75.1 18.2 56.1 29.9 1136062.1 AD- 87.6 7.4 63.4 14.6 72.7 15.4 69.6 9.6 1136063.1 AD- 77.7 11.2 91.6 7.0 152.9 110.5 99.5 32.0 1136064.1 AD- 64.0 13.4 80.7 20.6 81.6 22.8 72.8 5.9 1136065.1 AD- 45.0 9.3 93.1 22.7 91.3 24.0 74.5 11.0 1136066.1 AD- 82.1 15.8 71.3 9.4 111.2 40.5 65.7 7.6 1136067.1 AD- 59.5 7.1 65.8 7.8 46.3 3.4 59.5 9.9 1136068.1 AD- 70.5 11.2 110.1 34.4 93.5 9.2 116.6 15.6 1136069.1 AD- 72.7 14.9 97.8 10.1 123.2 9.0 119.7 19.3 1136070.1 AD- 89.3 29.7 147.2 32.6 116.4 9.6 120.2 22.4 1136071.1 AD- 95.5 10.2 119.5 39.2 126.8 29.4 122.5 5.6 1136072.1 AD- 87.5 9.9 109.6 20.4 129.3 13.0 129.0 17.8 1136073.1 AD- 79.1 16.2 120.6 3.2 109.1 10.5 130.8 13.1 1136074.1 AD- 82.2 18.7 96.5 22.5 105.9 9.6 126.9 18.4 1136075.1 AD- 91.2 18.0 85.6 27.6 85.6 12.4 110.3 23.2 1136076.1 AD- 74.5 16.0 99.0 10.9 104.5 14.7 104.4 13.8 1136077.1 AD- 100.1 9.6 130.6 11.9 113.5 15.7 135.4 21.0 1136078.1 AD- 101.0 14.8 143.2 10.1 127.3 15.3 140.5 24.4 1136079.1 AD- 96.1 19.2 146.9 13.4 135.5 16.3 117.8 19.4 1136080.1 AD- 83.7 28.2 82.3 18.0 115.2 19.9 106.3 10.7 1136081.1 AD- 72.0 11.5 97.0 19.0 94.7 23.0 101.9 10.1 1136082.1 AD- 69.3 10.0 115.8 28.1 101.8 10.0 113.2 9.7 1136083.1 AD- 81.1 10.4 94.1 15.0 85.7 4.8 120.1 29.3 1136084.1 AD- 85.9 15.2 118.5 28.7 96.3 12.8 107.9 6.7 1136085.1 AD- 83.1 19.4 129.9 14.1 141.3 1.7 119.5 25.6 1136086.1 AD- 100.3 8.6 137.9 31.5 118.5 23.2 105.9 21.7 1136087.1 AD- 97.3 20.8 140.1 6.0 113.2 25.0 95.9 7.5 1136088.1 AD- 98.5 16.6 121.8 27.9 101.3 13.0 94.3 31.5 1136089.1 AD- 106.2 20.8 140.4 15.5 101.2 11.2 99.1 9.0 1136090.1 AD- 73.4 7.7 96.1 17.9 91.4 11.8 107.2 18.0 1136091.1 AD- 72.0 15.5 107.6 7.0 94.3 10.6 100.8 19.5 1136092.1 AD- 85.7 19.5 105.7 25.1 126.6 14.6 122.6 27.1 1136093.1 AD- 82.4 14.4 141.4 17.8 128.2 4.1 143.9 43.9 1136094.1 AD- 85.0 16.7 112.5 26.9 118.6 13.7 97.8 25.1 1136095.1 AD- 104.5 14.0 119.3 28.0 113.7 15.1 111.6 8.6 1136096.1 AD- 94.4 18.7 121.0 28.7 99.6 22.6 81.0 7.2 1136097.1 AD- 66.1 19.4 100.5 41.4 79.1 15.3 70.6 7.3 1136098.1 AD- 68.3 4.4 88.1 9.5 111.4 7.6 105.9 10.8 1136099.1 AD- 64.9 19.9 99.2 3.7 112.4 17.2 123.6 20.1 1136100.1 AD- 67.7 12.8 107.3 26.3 121.3 16.0 117.7 18.2 1136101.1 AD- 86.3 24.3 132.6 11.5 138.2 14.7 144.2 15.2 1136102.1 AD- 81.7 18.6 121.1 32.7 100.0 19.9 124.6 27.2 1136103.1 AD- 69.6 6.9 101.7 5.5 106.3 12.3 99.0 14.6 1136104.1 AD- 83.9 26.6 96.0 28.4 88.3 6.1 95.9 16.0 1136105.1 AD- 74.4 18.2 121.1 39.6 73.2 10.2 97.2 13.7 1136106.1 AD- 69.3 9.2 102.7 12.1 78.9 11.3 74.4 3.5 1136107.1 AD- 69.5 9.8 105.9 9.3 96.8 19.0 111.2 8.6 1136108.1 AD- 72.8 11.1 111.8 12.9 114.2 8.7 122.4 20.9 1136109.1 AD- 100.8 9.3 128.7 27.3 125.8 17.6 124.7 17.2 1136110.1 AD- 94.8 10.0 95.0 18.0 104.9 15.7 100.7 15.9 1136111.1 AD- 82.1 10.6 113.0 19.4 84.3 17.3 114.3 18.2 1136112.1 AD- 56.6 8.9 101.7 21.4 90.1 5.0 98.0 36.5 1136114.1 AD- 72.9 15.9 121.0 29.4 116.4 21.1 95.7 12.5 1136115.1 AD- 82.9 5.1 110.2 7.2 100.3 8.4 88.8 11.4 1136116.1 AD- 63.2 26.4 97.7 4.8 119.2 27.4 90.4 13.0 1136117.1 AD- 61.6 4.3 95.5 25.6 84.9 10.6 86.1 11.7 1136118.1 AD- 82.1 15.1 116.6 5.7 93.1 17.2 95.9 8.6 1136119.1 AD- 74.9 9.7 93.3 18.1 77.3 10.4 92.8 11.7 1136120.1 AD- 83.0 20.0 69.1 21.8 65.5 6.7 73.5 8.7 1136121.1 AD- 68.4 10.0 90.0 11.9 97.5 12.8 90.8 8.0 1136122.1 AD- 76.3 14.8 109.2 6.3 108.2 19.9 85.4 25.2 1136123.1 AD- 76.1 9.4 97.8 18.2 98.9 14.5 96.9 16.9 1136124.1 AD- 77.7 21.2 100.9 17.9 115.5 27.9 109.8 18.2 1136125.1 AD- 91.7 16.1 92.7 13.9 104.3 27.1 101.7 11.3 1136126.1 AD- 58.6 8.7 82.4 24.5 88.4 17.5 83.0 11.3 1136127.1 AD- 76.2 22.3 99.3 18.3 92.0 9.4 81.3 13.0 1136128.1 AD- 68.2 14.1 68.7 22.7 71.5 17.4 66.4 15.3 1136129.1 AD- 80.8 13.1 97.9 6.2 97.0 11.2 76.9 7.8 1136130.1 AD- 76.6 19.3 113.4 2.7 103.2 26.0 89.0 8.9 1136131.1 AD- 78.2 9.9 123.5 16.4 132.2 21.3 110.4 19.0 1136132.1 AD- 84.8 19.3 87.9 18.4 108.8 23.8 101.2 7.4 1136133.1 AD- 79.4 5.0 94.6 5.1 96.5 15.0 110.6 21.3 1136134.1 AD- 87.2 14.7 75.4 25.0 90.2 11.1 79.6 25.2 1136135.1 AD- 65.1 6.8 55.1 31.4 69.7 12.2 63.4 15.5 1136136.1 AD- 83.6 9.9 107.0 0.5 102.7 17.4 96.2 6.1 1136137.1 AD- 59.1 7.9 116.3 22.9 92.4 6.3 110.3 12.6 1136138.1 AD- 80.1 7.5 102.2 15.4 123.0 15.0 113.0 26.8 1136139.1 AD- 77.9 15.8 73.4 17.7 107.6 12.2 103.5 24.3 1136140.1 AD- 63.5 7.4 73.2 15.7 78.5 10.8 96.1 16.1 1136141.1 AD- 72.4 15.8 63.2 11.8 76.8 14.6 73.9 22.0 1136142.1 AD- 69.1 6.8 63.6 11.9 68.0 17.8 64.6 9.4 1136143.1 AD- 80.1 8.4 75.1 10.5 99.9 19.3 83.1 4.2 1136144.1 AD- 83.3 5.0 97.0 21.2 96.8 9.8 97.4 19.6 1136145.1 AD- 94.1 16.7 104.8 32.0 95.2 23.4 97.1 15.9 1136146.1 AD- 97.9 8.6 112.2 15.3 114.9 7.5 100.2 10.3 1136147.1 AD- 86.8 11.6 72.8 24.6 95.8 12.9 100.1 20.1 1136148.1 AD- 81.9 12.9 85.2 10.2 90.9 17.0 97.5 34.3 1136149.1 AD- 71.1 5.3 80.7 16.4 87.1 12.8 94.7 34.4 1136150.1 AD- 89.7 19.7 63.0 38.6 67.7 3.9 62.1 22.2 1136151.1 AD- 84.8 17.6 93.9 21.0 105.4 14.2 67.3 8.3 1136152.1 AD- 80.9 9.8 105.6 2.5 101.6 8.9 87.8 12.2 1136153.1 AD- 86.4 7.8 83.5 15.1 103.4 9.4 97.1 10.3 1136154.1 AD- 90.9 23.6 112.1 16.9 109.5 7.9 88.2 6.4 1136155.1 AD- 69.2 12.9 82.9 26.6 93.4 9.8 88.3 12.9 1136156.1 AD- 76.5 9.5 75.5 16.4 96.3 12.5 82.6 10.7 1136157.1 AD- 78.1 10.3 85.5 6.5 88.7 7.4 71.2 9.2 1136158.1 AD- 43.3 40.2 121.4 41.6 109.5 11.3 143.7 73.3 1136159.1 AD- 95.8 25.4 118.1 53.7 115.4 13.3 130.2 33.1 1136160.1 AD- 156.0 58.2 158.0 40.7 114.7 14.2 132.5 23.9 1136161.1 AD- 69.3 23.9 120.9 33.9 119.8 12.2 160.5 24.0 1136162.1 AD- 74.1 4.3 94.9 15.8 101.1 19.6 107.3 18.3 1136163.1 AD- 102.0 13.5 162.8 81.5 97.7 33.3 128.5 10.1 1136164.1 AD- 78.9 28.8 144.5 32.9 92.1 21.7 134.9 48.1 1136165.1 AD- 75.5 34.6 129.8 26.6 75.9 136.2 54.8 1136166.1 AD- 168.2 35.6 110.6 17.3 98.8 47.9 77.2 42.5 1136167.1 AD- 107.5 25.5 98.5 14.6 132.1 8.3 124.0 24.5 1136168.1 AD- 77.7 14.0 93.4 13.3 87.9 24.6 99.9 8.8 1136169.1 AD- 87.2 23.0 113.2 28.8 115.9 4.3 145.8 19.7 1136170.1 AD- 88.8 28.1 141.1 2.8 125.2 33.6 140.4 6.1 1136171.1 AD- 101.1 18.3 106.3 21.5 106.1 29.3 135.0 23.7 1136172.1 AD- 89.1 26.0 105.4 20.8 96.5 17.9 88.9 10.9 1136173.1 AD- 98.9 17.1 56.1 33.7 94.9 17.2 122.7 64.3 1136174.1 AD- 119.8 18.5 130.4 31.7 73.5 23.3 129.4 10.1 1136175.1 AD- 95.1 17.7 132.3 10.0 128.0 17.0 125.7 17.9 1136176.1 AD- 111.2 12.0 129.7 18.6 130.1 30.2 109.7 6.2 1136177.1 AD- 81.9 12.8 111.7 15.8 120.8 18.5 124.3 49.9 1136178.1 AD- 103.0 13.1 123.6 10.8 129.2 12.7 94.3 10.2 1136179.1 AD- 94.1 14.4 119.8 20.6 109.1 8.7 104.5 5.6 1136180.1 AD- 80.4 8.6 61.1 4.8 103.7 32.6 98.9 20.2 1136181.1 AD- 146.5 24.7 113.3 14.7 124.3 60.8 117.5 3.7 1136182.1 AD- 81.8 16.0 114.8 23.7 130.1 19.2 129.3 19.0 1136183.1 AD- 100.5 10.5 140.0 26.6 119.3 27.8 109.2 18.4 1136184.1 AD- 86.3 13.5 113.6 20.7 137.7 7.9 129.4 27.5 1136185.1 AD- 68.2 10.7 114.0 3.0 130.5 24.9 104.8 11.0 1136186.1 AD- 85.9 20.0 97.3 17.9 103.1 18.0 91.6 4.3 1136187.1 AD- 87.2 6.2 126.8 6.8 103.7 17.8 95.3 25.2 1136188.1 AD- 87.1 34.4 138.2 21.0 126.3 18.3 122.3 27.9 1136189.1 AD- 116.7 40.3 121.6 10.3 134.5 15.8 163.3 48.0 1136190.1 AD- 105.2 15.4 130.1 34.0 124.4 16.8 121.0 27.5 1136191.1 AD- 97.3 17.1 151.8 11.8 131.1 30.0 145.5 16.0 1136192.1 AD- 93.6 21.5 110.8 20.2 130.3 34.5 116.7 25.5 1136193.1 AD- 91.0 17.6 125.2 22.1 126.9 7.4 103.6 23.3 1136194.1 AD- 109.6 21.9 92.9 19.4 108.0 13.5 81.9 12.5 1136195.1 AD- 85.2 33.9 85.2 27.3 104.2 20.9 100.2 10.9 1136196.1 AD- 104.7 12.5 86.5 17.1 127.6 39.6 132.2 40.1 1136197.1 AD- 122.2 24.1 103.0 19.9 138.2 18.1 124.0 34.7 1136198.1 AD- 76.7 17.3 95.9 7.4 116.7 12.3 111.4 12.1 1136199.1 AD- 91.6 16.2 123.1 25.2 126.9 32.2 104.0 36.0 1136200.1 AD- 73.6 15.4 99.6 26.1 83.8 11.3 71.5 19.1 1136201.1 AD- 72.7 8.6 95.9 24.1 114.6 28.7 87.1 17.2 1136202.1 AD- 69.9 11.0 74.6 18.4 126.8 20.6 85.4 20.3 1136203.1 AD- 99.9 53.1 89.2 16.7 137.2 4.5 138.9 4.8 1136204.1 AD- 73.4 17.8 107.7 17.5 144.7 21.8 136.0 38.7 1136205.1 AD- 87.3 18.9 120.5 11.0 107.3 20.5 115.5 40.3 1136206.1 AD- 85.5 21.6 112.6 26.2 81.9 28.3 74.5 4.6 1136207.1 AD- 83.6 5.3 115.1 15.6 79.9 26.1 80.1 7.7 1136208.1 AD- 74.1 16.9 83.9 7.0 101.2 33.8 68.2 18.6 1136209.1 AD- 79.2 15.2 92.9 17.0 92.2 25.5 80.1 12.7 1136210.1 AD- 77.9 18.3 75.0 8.5 110.5 6.5 80.6 6.9 1136211.1 AD- 76.2 41.5 109.6 24.4 118.0 16.6 139.1 3.9 1136212.1 AD- 95.4 20.3 92.9 11.9 123.4 16.0 109.1 16.9 1136213.1 AD- 62.4 14.1 76.6 6.7 89.2 17.3 102.8 27.1 1136214.1 AD- 63.4 16.7 105.3 15.1 100.4 34.7 73.6 25.3 1136215.1 AD- 66.8 17.6 82.2 29.4 127.5 22.5 108.9 16.2 1136216.1 AD- 60.3 20.1 63.7 10.4 64.0 13.9 61.2 27.6 1136217.1 AD- 66.6 13.3 60.5 8.7 70.1 14.6 65.6 16.3 1136218.1 AD- 65.6 9.5 107.3 32.2 95.2 19.9 75.7 3.7 1136219.1 AD- 111.3 22.6 129.8 14.7 135.3 32.2 155.5 85.4 1136220.1 AD- 85.1 10.8 101.0 8.3 102.0 23.9 105.4 25.9 1136221.1 AD- 85.7 2.2 108.6 13.3 74.7 29.6 60.8 13.5 1136222.1 AD- 76.1 23.3 65.4 13.2 76.0 30.7 79.3 17.7 1136223.1 AD- 66.5 6.4 62.6 14.6 47.6 6.1 48.1 11.1 1136224.1 AD- 67.1 7.3 51.4 20.4 48.0 11.6 55.6 13.0 1136225.1 AD- 59.5 6.6 68.7 10.4 96.1 19.1 78.7 6.3 1136226.1 AD- 107.2 4.1 125.4 21.5 80.0 13.7 112.2 74.8 1136227.1 AD- 87.8 35.4 104.9 2.0 102.5 28.2 94.9 16.3 1136228.1 AD- 74.8 21.7 70.0 10.9 58.6 14.0 52.2 1.6 1136229.1 AD- 60.7 17.2 77.6 6.4 54.3 5.5 48.5 8.1 1136230.1 AD- 60.2 15.3 64.9 17.5 62.8 25.9 39.2 8.6 1136231.1 AD- 53.8 19.5 49.8 7.0 71.1 15.0 64.2 12.0 1136232.1 AD- 67.8 10.5 75.0 16.4 99.8 9.7 89.5 18.3 1136233.1 AD- 135.9 39.0 99.5 7.3 76.8 6.8 86.4 32.5 1136234.1 AD- 106.5 18.7 97.4 17.9 92.2 11.3 81.5 18.2 1136235.1 AD- 73.8 11.1 83.1 7.2 85.4 12.0 81.7 5.4 1136236.1 AD- 72.0 45.6 79.6 5.5 58.9 17.0 52.5 7.6 1136237.1 AD- 73.1 3.2 75.4 12.1 52.1 4.1 45.3 7.1 1136238.1 AD- 59.5 17.2 66.6 18.3 57.2 8.8 58.9 27.6 1136239.1 AD- 61.1 7.6 63.8 2.7 89.7 21.7 75.9 6.2 1136240.1 AD- 93.9 37.4 81.2 7.6 74.3 19.1 131.9 66.6 1136241.1 AD- 119.9 28.2 142.2 79.5 94.5 17.8 89.0 65.8 1136242.1 AD- 81.2 2.4 69.7 15.0 94.7 32.0 109.6 46.7 1136243.1 AD- 66.9 32.5 66.8 3.3 67.7 11.6 53.6 1.6 1136244.1 AD- 89.6 25.0 85.4 14.5 79.9 32.3 54.9 11.8 1136245.1 AD- 70.6 9.3 67.8 7.7 69.2 18.8 47.9 14.6 1136246.1 AD- 99.4 30.6 69.9 11.6 51.5 14.4 64.9 23.9 1136247.1 AD- 68.2 10.9 66.0 7.4 82.0 32.8 62.3 9.7 1136248.1 AD- 137.1 53.3 111.3 46.1 66.0 44.0 101.4 105.9 1136249.1 AD- 112.9 11.1 143.8 8.3 121.6 19.0 104.3 22.7 1136250.1 AD- 108.3 30.3 149.1 26.0 135.9 15.9 160.4 106.8 1136251.1 AD- 94.4 5.2 127.0 37.2 125.2 5.1 97.7 35.3 1136252.1 AD- 112.3 16.8 130.1 14.8 127.2 23.0 182.7 108.0 1136253.1 AD- 148.8 15.9 129.8 30.2 140.8 26.9 82.2 50.6 1136254.1 AD- 103.0 20.9 119.4 32.1 141.8 42.9 90.3 54.0 1136255.1 AD- 124.3 41.8 108.0 22.9 89.3 27.9 118.4 27.6 1136256.1 AD- 123.8 50.0 97.2 20.6 104.0 6.6 52.4 7.6 1136257.1 AD- 61.2 10.2 124.6 23.8 118.5 24.8 117.7 16.9 1136258.1 AD- 89.2 26.4 120.0 20.2 143.7 13.9 98.8 5.8 1136259.1 AD- 93.3 22.2 135.4 22.6 152.5 34.4 150.5 27.6 1136260.1 AD- 80.6 17.3 111.9 13.8 125.0 33.0 127.6 29.7 1136261.1 AD- 71.8 12.8 113.2 7.6 133.9 2.7 104.6 9.0 1136262.1 AD- 81.0 20.7 93.6 19.1 105.3 24.0 101.2 8.5 1136263.1 AD- 73.3 13.3 82.8 20.6 85.6 8.3 N/A N/A 1136264.1 AD- 102.0 12.2 122.2 19.5 103.1 11.1 N/A N/A 1136265.1 AD- 95.9 22.1 117.0 27.4 120.9 15.0 100.2 22.0 1136266.1 AD- 102.3 18.1 149.1 13.2 156.4 45.6 197.8 80.6 1136267.1 AD- 95.8 20.1 161.8 23.0 96.2 20.3 N/A N/A 1136268.1 AD- 83.5 12.5 129.0 9.1 113.2 17.3 153.8 51.2 1136269.1 AD- 116.6 19.9 112.3 10.5 115.9 21.3 91.9 27.5 1136270.1 AD- 82.9 24.6 81.1 12.3 68.2 20.5 67.0 28.6 1136271.1 AD- 71.3 14.3 86.2 8.9 94.2 15.6 78.1 10.3 1136272.1 AD- 77.6 16.4 125.1 10.3 129.3 21.4 107.4 17.7 1136273.1 AD- 96.3 30.6 169.2 39.0 104.4 19.2 333.6 177.6 1136274.1 AD- 103.6 13.0 146.3 17.2 97.7 21.7 N/A N/A 1136275.1 AD- 101.7 13.3 143.9 30.2 133.7 27.5 129.2 42.3 1136276.1 AD- 82.3 22.6 103.5 6.1 106.7 33.3 80.3 17.4 1136277.1 AD- 69.5 29.9 93.6 24.6 85.4 23.3 72.6 22.4 1136278.1 AD- 64.1 9.0 94.7 12.9 78.5 33.4 76.7 31.1 1136279.1 AD- 74.3 22.3 107.2 15.8 108.0 23.7 85.2 28.9 1136280.1 AD- 123.8 23.0 148.2 30.4 117.0 11.9 135.4 32.4 1136281.1 AD- 86.4 24.8 147.3 39.3 153.6 19.2 257.4 178.0 1136282.1 AD- 63.2 20.2 85.5 26.9 110.6 32.6 N/A N/A 1136283.1 AD- 84.0 14.1 114.3 11.9 77.9 18.0 208.9 84.2 1136284.1 AD- 88.8 16.1 93.0 29.4 103.1 17.1 82.9 20.6 1136285.1 AD- 66.0 10.5 59.8 8.7 51.2 30.0 60.3 14.0 1136286.1 AD- 76.6 25.4 97.0 12.8 89.7 14.3 145.0 110.3 1136287.1 AD- 73.0 23.3 99.8 28.2 107.0 10.2 86.6 11.2 1136288.1 AD- 79.0 9.3 104.0 20.5 144.6 8.6 141.6 41.6 1136289.1 AD- 75.1 17.2 117.4 20.4 80.4 14.3 N/A N/A 1136290.1 AD- 50.6 10.3 73.9 19.4 80.5 34.1 N/A N/A 1136291.1 AD- 65.8 10.9 93.5 14.3 84.7 26.4 113.4 37.8 1136292.1 AD- 78.3 7.4 82.1 9.9 65.6 20.3 71.4 17.4 1136293.1 AD- 59.4 7.5 81.6 12.2 62.3 38.5 114.0 82.5 1136294.1 AD- 68.9 9.1 98.9 19.2 115.6 12.7 93.5 16.7 1136295.1 AD- 85.6 23.0 121.5 13.0 91.4 14.3 107.7 15.6 1136296.1 AD- 92.2 24.2 126.8 24.1 73.8 13.7 218.7 44.7 1136297.1 AD- 89.7 11.3 91.1 3.2 75.8 11.0 N/A N/A 1136298.1 AD- 75.7 4.3 112.6 26.8 76.8 9.4 98.1 10.5 1136299.1 AD- 79.9 23.9 99.6 13.2 85.8 28.4 77.2 1.8 1136300.1 AD- 81.3 16.5 58.9 10.8 75.2 18.8 52.9 10.3 1136301.1 AD- 69.9 22.1 102.0 40.8 70.5 23.3 73.7 9.2 1136302.1 AD- 73.6 20.6 92.2 16.0 104.6 26.4 103.9 6.0 1136303.1 AD- 80.4 13.2 108.2 16.5 102.0 12.2 106.9 40.0 1136304.1 AD- 79.2 12.9 114.0 17.1 105.6 28.9 N/A N/A 1136305.1 AD- 78.0 6.6 111.2 15.3 100.5 23.9 N/A N/A 1136306.1 AD- 76.1 12.7 115.6 28.5 83.8 15.3 96.8 21.5 1136307.1 AD- 67.4 17.1 66.7 21.3 63.3 17.1 67.4 5.6 1136308.1 AD- 65.1 26.6 79.8 11.5 74.5 4.1 60.7 23.8 1136309.1 AD- 71.3 22.6 76.1 16.3 108.9 35.2 71.2 25.5 1136310.1 AD- 59.3 9.8 94.5 8.0 96.2 14.4 89.3 17.7 1136311.1 AD- 63.7 6.0 105.0 12.9 90.5 5.7 89.3 11.6 1136312.1 AD- 75.7 3.9 105.1 37.5 89.8 21.6 88.8 19.0 1136313.1 AD- 108.8 17.9 95.8 12.0 83.8 21.1 87.0 23.5 1136314.1 AD- 92.0 15.8 83.7 15.5 77.1 13.5 73.5 18.1 1136315.1 AD- 85.1 16.3 66.7 10.9 60.0 8.0 49.3 11.8 1136316.1 AD- 64.5 7.9 72.5 16.7 89.9 21.4 77.8 9.4 1136317.1 AD- 88.2 12.5 86.4 13.6 91.0 17.1 105.4 24.8 1136318.1 AD- 63.9 18.5 120.7 38.2 119.4 20.1 92.4 19.3 1136319.1 AD- 68.9 15.0 100.9 9.9 105.8 27.7 89.6 9.0 1136320.1 AD- 96.1 24.6 91.0 32.8 90.1 16.5 73.3 9.0 1136321.1 AD- 101.9 24.1 80.8 18.8 63.5 11.5 71.3 20.1 1136322.1 AD- 73.2 15.8 62.6 22.8 61.0 18.6 68.1 27.7 1136323.1 AD- 110.1 12.5 88.6 18.2 87.0 9.3 63.6 11.8 1136324.1 AD- 83.4 16.1 90.3 7.4 81.7 17.5 65.5 25.1 1136325.1 AD- 87.7 28.8 101.2 12.0 70.3 15.4 59.2 11.4 1136326.1 AD- 142.0 57.3 137.3 10.5 63.4 8.7 85.7 13.0 1136327.1 AD- 90.4 10.6 85.2 11.8 91.7 31.9 63.8 21.7 1136328.1 AD- 79.2 17.1 89.8 26.0 49.4 9.6 52.0 7.9 1136329.1 AD- 125.2 36.0 78.1 21.5 76.5 9.9 67.1 22.8 1136330.1 AD- 100.0 34.1 81.0 2.9 86.7 13.1 64.3 25.9 1136331.1 AD- 106.9 22.8 72.0 38.2 69.4 23.1 54.3 20.7 1136332.1 AD- 76.4 29.8 110.9 45.1 61.2 8.1 59.3 14.5 1136333.1 AD- 105.3 58.3 93.3 14.6 49.5 19.3 47.3 21.0 1136334.1 AD- 71.5 11.5 113.4 24.5 76.3 28.7 70.0 40.8 1136335.1 AD- 136.1 69.8 104.1 23.7 81.2 23.9 57.7 18.1 1136336.1 AD- 87.1 35.7 100.8 20.0 59.4 28.2 38.8 8.9 1136337.1 AD- 124.5 31.0 86.8 14.0 51.3 4.8 77.9 75.1 1136338.1

Example 3. Additional Duplexes Targeting XDH

Additional duplexes targeting the xanthine dehydrogenase (XDH) gene, (human: NCBI refseqID NM_000379; NCBI GeneID: 7498) were designed using custom R and Python scripts. The human NM_000379 REFSEQ mRNA, version 4, has a length of 5715 bases.

siRNAs were synthesized and annealed using routine methods known in the art and described above.

Detailed lists of the unmodified XDH sense and antisense strand nucleotide sequences are shown in Table 6. Detailed lists of the modified XDH sense and antisense strand nucleotide sequences are shown in Table 7.

The additional duplexes for screened for activity in vitro.

Free uptake experiments were performed by adding 2.5 μl of siRNA duplexes in PBS per well into a 96 well plate. Complete growth media (47.5 μl) containing about 1.5×10⁴ PMH or PHH was then added to the siRNA. Cells were incubated for 48 hours prior to RNA purification and RT-qPCR. Single dose experiments in PHH were performed at 500 nM, 100 nM, 10 nM and/or 0.1 nM in PHH and in PMH at 500 nM, 100 nM, nM, and 1 nM final duplex concentration.

For transfection, PHH cells were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in Eagle's Minimum Essential Medium (Gibco) supplemented with 10% FBS (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 7.5 μl of Opti-MEM plus 0.1 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 2.5 μl of each siRNA duplex to an individual well in a 384-well plate. The mixture was then incubated at room temperature for 15 minutes. Forty μl of complete growth media without antibiotic containing ˜1.5×10⁴ PHH cells were then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed at 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM, 0.0064 nM, 0.00128 nM and 0.0001256 nM final duplex concentration.

Total RNA isolation was performed using DYNABEADS. Briefly, cells were lysed in 10 μl of Lysis/Binding Buffer containing 3 μL of beads per well and mixed for 10 minutes on an electrostatic shaker. The washing steps were automated on a Biotek EL406, using a magnetic plate support. Beads were washed (in 3 μL) once in Buffer A, once in Buffer B, and twice in Buffer E, with aspiration steps in between. Following a final aspiration, complete 12 μL RT mixture was added to each well, as described below.

For cDNA synthesis, a master mix of 1.5 μl 10× Buffer, 0.6 μl 10×dNTPs, 1.5 μl Random primers, 0.75 μl Reverse Transcriptase, 0.75 μl RNase inhibitor and 9.9 μl of H₂O per reaction were added per well. Plates were sealed, agitated for 10 minutes on an electrostatic shaker, and then incubated at 37 degrees C. for 2 hours. Following this, the plates were agitated at 80 degrees C. for 8 minutes.

RT-qPCR was performed as described above and relative fold change was calculated as described above.

The results of the free uptake experiments of the dsRNA agents listed in Tables 6 and 7 in PHH are shown in Tables 8 and 9. The results of the transfection experiments of the dsRNA agents listed in Tables 6 and 7 in PHH are shown in Tables 10-12. The IC₅₀ values for the dsRNA agents from Tables 10-12 are included in Table 13.

TABLE 6 Unmodified Sense and Antisense Strand Sequences of Xanthine Dehydrogenase dsRNA Agents SEQ SEQ ID Range in ID Range in Duplex Name Sense Sequence 5′ to 3′ NO: NM_000379.4 Antisense Sequence 5′ to 3′ NO: NM_000379.4 AD-1135990.2 GCACAGUGAUGCUCUCCAAGU 34 228-248 ACUUGGAGAGCAUCACUGUGCAA 393 226-248 AD-1135991.2 CACAGUGAUGCUCUCCAAGUU 35 229-249 AACUTGGAGAGCAUCACUGUGCA 394 227-249 AD-1297597.1 ACAGUGAUGCUCUCCAAGUAU 1818 230-250 AUACTUGGAGAGCAUCACUGUGC 1934 228-250 AD-1297598.1 CAGUGAUGCUCUCCAAGUAUU 1819 231-251 AAUACUUGGAGAGCAUCACUGUG 1935 229-251 AD-1297599.1 AGUGAUGCUCUCCAAGUAUGU 1820 232-252 ACAUACUUGGAGAGCAUCACUGU 1936 230-252 AD-1297600.1 GUGAUGCUCUCCAAGUAUGAU 1821 233-253 AUCAUACUUGGAGAGCAUCACUG 1937 231-253 AD-1297601.1 UGAUGCUCUCCAAGUAUGAUU 1822 234-254 AAUCAUACUUGGAGAGCAUCACU 1938 232-254 AD-1135992.2 GAUGCUCUCCAAGUAUGAUCU 36 235-255 AGAUCAUACUUGGAGAGCAUCAC 395 233-255 AD-1135993.2 AUGCUCUCCAAGUAUGAUCGU 37 236-256 ACGAUCAUACUUGGAGAGCAUCA 396 234-256 AD-1135994.2 UGCUCUCCAAGUAUGAUCGUU 38 237-257 AACGAUCAUACUUGGAGAGCAUC 397 235-257 AD-1135995.2 GCUCUCCAAGUAUGAUCGUCU 39 238-258 AGACGAUCAUACUUGGAGAGCAU 398 236-258 AD-1135996.2 CUCUCCAAGUAUGAUCGUCUU 40 239-259 AAGACGAUCAUACUUGGAGAGCA 399 237-259 AD-1297602.1 UCUCCAAGUAUGAUCGUCUGU 1823 240-260 ACAGACGAUCAUACUUGGAGAGC 1939 238-260 AD-1297603.1 CUCCAAGUAUGAUCGUCUGCU 1824 241-261 AGCAGACGAUCAUACUUGGAGAG 1940 239-261 AD-1297604.1 UCCAAGUAUGAUCGUCUGCAU 1825 242-262 AUGCAGACGAUCAUACUUGGAGA 1941 240-262 AD-1297605.1 CCAAGUAUGAUCGUCUGCAGU 1826 243-263 ACUGCAGACGAUCAUACUUGGAG 1942 241-263 AD-1297606.1 CAAGUAUGAUCGUCUGCAGAU 1827 244-264 AUCUGCAGACGAUCAUACUUGGA 1943 242-264 AD-1297607.1 AAGUAUGAUCGUCUGCAGAAU 1828 245-265 AUUCTGCAGACGAUCAUACUUGG 1944 243-265 AD-1297608.1 AGUAUGAUCGUCUGCAGAACU 1829 246-266 AGUUCUGCAGACGAUCAUACUUG 1945 244-266 AD-1297609.1 GUAUGAUCGUCUGCAGAACAU 1830 247-267 AUGUTCTGCAGACGAUCAUACUU 1946 245-267 AD-1297610.1 UAUGAUCGUCUGCAGAACAAU 1831 248-268 AUUGTUCUGCAGACGAUCAUACU 1947 246-268 AD-1297611.1 AUGAUCGUCUGCAGAACAAGU 1832 249-269 ACUUGUUCUGCAGACGAUCAUAC 1948 247-269 AD-1136037.2 GGGAGUAUUUCUCAGCAUUCU 81 1320-1340 AGAATGCUGAGAAAUACUCCCCC 440 1318-1340 AD-1136038.2 GGAGUAUUUCUCAGCAUUCAU 82 1321-1341 AUGAAUGCUGAGAAAUACUCCCC 441 1319-1341 AD-1136039.2 GAGUAUUUCUCAGCAUUCAAU 83 1322-1342 AUUGAAUGCUGAGAAAUACUCCC 442 1320-1342 AD-1136040.2 AGUAUUUCUCAGCAUUCAAGU 84 1323-1343 ACUUGAAUGCUGAGAAAUACUCC 443 1321-1343 AD-1136041.2 GUAUUUCUCAGCAUUCAAGCU 85 1324-1344 AGCUTGAAUGCUGAGAAAUACUC 444 1322-1344 AD-1297612.1 UAUUUCUCAGCAUUCAAGCAU 1833 1325-1345 AUGCTUGAAUGCUGAGAAAUACU 1949 1323-1345 AD-1297613.1 AUUUCUCAGCAUUCAAGCAGU 1834 1326-1346 ACUGCUUGAAUGCUGAGAAAUAC 1950 1324-1346 AD-1297614.1 UUUCUCAGCAUUCAAGCAGGU 1835 1327-1347 ACCUGCTUGAAUGCUGAGAAAUA 1951 1325-1347 AD-1297615.1 UUCUCAGCAUUCAAGCAGGCU 1836 1328-1348 AGCCTGCUUGAAUGCUGAGAAAU 1952 1326-1348 AD-1297616.1 UCUCAGCAUUCAAGCAGGCCU 1837 1329-1349 AGGCCUGCUUGAAUGCUGAGAAA 1953 1327-1349 AD-1297617.1 CUCAGCAUUCAAGCAGGCCUU 1838 1330-1350 AAGGCCTGCUUGAAUGCUGAGAA 1954 1328-1350 AD-1297618.1 UCAGCAUUCAAGCAGGCCUCU 1839 1331-1351 AGAGGCCUGCUUGAAUGCUGAGA 1955 1329-1351 AD-1297619.1 CAGCAUUCAAGCAGGCCUCCU 1840 1332-1352 AGGAGGCCUGCUUGAAUGCUGAG 1956 1330-1352 AD-1297620.1 AAGAAGGUUCCAGGGUUUGUU 1841 1955-1975 AACAAACCCUGGAACCUUCUUAG 1957 1953-1975 AD-1297621.1 AGAAGGUUCCAGGGUUUGUUU 1842 1956-1976 AAACAAACCCUGGAACCUUCUUA 1958 1954-1976 AD-1297622.1 GAAGGUUCCAGGGUUUGUUUU 1843 1957-1977 AAAACAAACCCUGGAACCUUCUU 1959 1955-1977 AD-1297623.1 AAGGUUCCAGGGUUUGUUUGU 1844 1958-1978 ACAAACAAACCCUGGAACCUUCU 1960 1956-1978 AD-1297624.1 AGGUUCCAGGGUUUGUUUGUU 1845 1959-1979 AACAAACAAACCCUGGAACCUUC 1961 1957-1979 AD-1297625.1 GGUUCCAGGGUUUGUUUGUUU 1846 1960-1980 AAACAAACAAACCCUGGAACCUU 1962 1958-1980 AD-1297626.1 GUUCCAGGGUUUGUUUGUUUU 1847 1961-1981 AAAACAAACAAACCCUGGAACCU 1963 1959-1981 AD-1136050.2 UUCCAGGGUUUGUUUGUUUCU 94 1962-1982 AGAAACAAACAAACCCUGGAACC 453 1960-1982 AD-1297627.1 UCCAGGGUUUGUUUGUUUCAU 1848 1963-1983 AUGAAACAAACAAACCCUGGAAC 1964 1961-1983 AD-1297628.1 CCAGGGUUUGUUUGUUUCAUU 1849 1964-1984 AAUGAAACAAACAAACCCUGGAA 1965 1962-1984 AD-1136051.2 CAGGGUUUGUUUGUUUCAUUU 95 1965-1985 AAAUGAAACAAACAAACCCUGGA 454 1963-1985 AD-1136052.2 GGGUUUGUUUGUUUCAUUUCU 96 1967-1987 AGAAAUGAAACAAACAAACCCUG 455 1965-1987 AD-1136053.2 GGUUUGUUUGUUUCAUUUCCU 97 1968-1988 AGGAAAUGAAACAAACAAACCCU 456 1966-1988 AD-1297629.1 GUUUGUUUGUUUCAUUUCCGU 1850 1969-1989 ACGGAAAUGAAACAAACAAACCC 1966 1967-1989 AD-1297630.1 UUUGUUUGUUUCAUUUCCGCU 1851 1970-1990 AGCGGAAAUGAAACAAACAAACC 1967 1968-1990 AD-1136054.2 UUGUUUGUUUCAUUUCCGCUU 98 1971-1991 AAGCGGAAAUGAAACAAACAAAC 457 1969-1991 AD-1297631.1 UGUUUGUUUCAUUUCCGCUGU 1852 1972-1992 ACAGCGGAAAUGAAACAAACAAA 1968 1970-1992 AD-1297632.1 GUUUGUUUCAUUUCCGCUGAU 1853 1973-1993 AUCAGCGGAAAUGAAACAAACAA 1969 1971-1993 AD-1297633.1 UUUGUUUCAUUUCCGCUGAUU 1854 1974-1994 AAUCAGCGGAAAUGAAACAAACA 1970 1972-1994 AD-1297634.1 UUGUUUCAUUUCCGCUGAUGU 1855 1975-1995 ACAUCAGCGGAAAUGAAACAAAC 1971 1973-1995 AD-1297635.1 UGUUUCAUUUCCGCUGAUGAU 1856 1976-1996 AUCATCAGCGGAAAUGAAACAAA 1972 1974-1996 AD-1297636.1 GUUUCAUUUCCGCUGAUGAUU 1857 1977-1997 AAUCAUCAGCGGAAAUGAAACAA 1973 1975-1997 AD-1297637.1 UUUCAUUUCCGCUGAUGAUGU 1858 1978-1998 ACAUCAUCAGCGGAAAUGAAACA 1974 1976-1998 AD-1297638.1 GGAGAUGGAGCUCUUUGUGUU 1859 2353-2373 AACACAAAGAGCUCCAUCUCCCC 1975 2351-2373 AD-1297639.1 GAGAUGGAGCUCUUUGUGUCU 1860 2354-2374 AGACACAAAGAGCUCCAUCUCCC 1976 2352-2374 AD-1297640.1 AGAUGGAGCUCUUUGUGUCUU 1861 2355-2375 AAGACACAAAGAGCUCCAUCUCC 1977 2353-2375 AD-1297641.1 GAUGGAGCUCUUUGUGUCUAU 1862 2356-2376 AUAGACACAAAGAGCUCCAUCUC 1978 2354-2376 AD-1297642.1 AUGGAGCUCUUUGUGUCUACU 1863 2357-2377 AGUAGACACAAAGAGCUCCAUCU 1979 2355-2377 AD-1297643.1 UGGAGCUCUUUGUGUCUACAU 1864 2358-2378 AUGUAGACACAAAGAGCUCCAUC 1980 2356-2378 AD-1297644.1 GGAGCUCUUUGUGUCUACACU 1865 2359-2379 AGUGTAGACACAAAGAGCUCCAU 1981 2357-2379 AD-1297645.1 GAGCUCUUUGUGUCUACACAU 1866 2360-2380 AUGUGUAGACACAAAGAGCUCCA 1982 2358-2380 AD-1136073.2 AGCUCUUUGUGUCUACACAGU 117 2361-2381 ACUGTGTAGACACAAAGAGCUCC 476 2359-2381 AD-1136074.2 GCUCUUUGUGUCUACACAGAU 118 2362-2382 AUCUGUGUAGACACAAAGAGCUC 477 2360-2382 AD-1136075.2 CUCUUUGUGUCUACACAGAAU 119 2363-2383 AUUCTGTGUAGACACAAAGAGCU 478 2361-2383 AD-1136076.2 UCUUUGUGUCUACACAGAACU 120 2364-2384 AGUUCUGUGUAGACACAAAGAGC 479 2362-2384 AD-1136077.2 CUUUGUGUCUACACAGAACAU 121 2365-2385 AUGUTCTGUGUAGACACAAAGAG 480 2363-2385 AD-1136078.2 UUUGUGUCUACACAGAACACU 122 2366-2386 AGUGTUCUGUGUAGACACAAAGA 481 2364-2386 AD-1297646.1 UUGUGUCUACACAGAACACCU 1867 2367-2387 AGGUGUTCUGUGUAGACACAAAG 1983 2365-2387 AD-1297647.1 UGUGUCUACACAGAACACCAU 1868 2368-2388 AUGGTGTUCUGUGUAGACACAAA 1984 2366-2388 AD-1297648.1 GUGUCUACACAGAACACCAUU 1869 2369-2389 AAUGGUGUUCUGUGUAGACACAA 1985 2367-2389 AD-1297649.1 UGUCUACACAGAACACCAUGU 1870 2370-2390 ACAUGGTGUUCUGUGUAGACACA 1986 2368-2390 AD-1297650.1 GUCUACACAGAACACCAUGAU 1871 2371-2391 AUCATGGUGUUCUGUGUAGACAC 1987 2369-2391 AD-1297651.1 UCUACACAGAACACCAUGAAU 1872 2372-2392 AUUCAUGGUGUUCUGUGUAGACA 1988 2370-2392 AD-1297652.1 CUACACAGAACACCAUGAAGU 1873 2373-2393 ACUUCAUGGUGUUCUGUGUAGAC 1989 2371-2393 AD-1297653.1 UACACAGAACACCAUGAAGAU 1874 2374-2394 AUCUTCAUGGUGUUCUGUGUAGA 1990 2372-2394 AD-1297654.1 GGGAACACCCAGGAUCUCUCU 1875 2681-2701 AGAGAGAUCCUGGGUGUUCCCCA 1991 2679-2701 AD-1297655.1 GGAACACCCAGGAUCUCUCUU 1876 2682-2702 AAGAGAGAUCCUGGGUGUUCCCC 1992 2680-2702 AD-1297656.1 GAACACCCAGGAUCUCUCUCU 1877 2683-2703 AGAGAGAGAUCCUGGGUGUUCCC 1993 2681-2703 AD-1297657.1 AACACCCAGGAUCUCUCUCAU 1878 2684-2704 AUGAGAGAGAUCCUGGGUGUUCC 1994 2682-2704 AD-1297658.1 ACACCCAGGAUCUCUCUCAGU 1879 2685-2705 ACUGAGAGAGAUCCUGGGUGUUC 1995 2683-2705 AD-1297659.1 CACCCAGGAUCUCUCUCAGAU 1880 2686-2706 AUCUGAGAGAGAUCCUGGGUGUU 1996 2684-2706 AD-1297660.1 ACCCAGGAUCUCUCUCAGAGU 1881 2687-2707 ACUCTGAGAGAGAUCCUGGGUGU 1997 2685-2707 AD-1297661.1 CCCAGGAUCUCUCUCAGAGUU 1882 2688-2708 AACUCUGAGAGAGAUCCUGGGUG 1998 2686-2708 AD-1297662.1 CCAGGAUCUCUCUCAGAGUAU 1883 2689-2709 AUACTCTGAGAGAGAUCCUGGGU 1999 2687-2709 AD-1297663.1 CAGGAUCUCUCUCAGAGUAUU 1884 2690-2710 AAUACUCUGAGAGAGAUCCUGGG 2000 2688-2710 AD-1136082.2 AGGAUCUCUCUCAGAGUAUUU 126 2691-2711 AAAUACTCUGAGAGAGAUCCUGG 485 2689-2711 AD-1136083.2 GGAUCUCUCUCAGAGUAUUAU 127 2692-2712 AUAATACUCUGAGAGAGAUCCUG 486 2690-2712 AD-1136084.2 GAUCUCUCUCAGAGUAUUAUU 128 2693-2713 AAUAAUACUCUGAGAGAGAUCCU 487 2691-2713 AD-1136085.2 AUCUCUCUCAGAGUAUUAUGU 129 2694-2714 ACAUAAUACUCUGAGAGAGAUCC 488 2692-2714 AD-1136086.2 UCUCUCUCAGAGUAUUAUGGU 130 2695-2715 ACCAUAAUACUCUGAGAGAGAUC 489 2693-2715 AD-1136087.2 CUCUCUCAGAGUAUUAUGGAU 131 2696-2716 AUCCAUAAUACUCUGAGAGAGAU 490 2694-2716 AD-1136088.2 UCUCUCAGAGUAUUAUGGAAU 132 2697-2717 AUUCCAUAAUACUCUGAGAGAGA 491 2695-2717 AD-1136089.2 CUCUCAGAGUAUUAUGGAACU 133 2698-2718 AGUUCCAUAAUACUCUGAGAGAG 492 2696-2718 AD-1136090.2 UCUCAGAGUAUUAUGGAACGU 134 2699-2719 ACGUUCCAUAAUACUCUGAGAGA 493 2697-2719 AD-1297664.1 CUCAGAGUAUUAUGGAACGAU 1885 2700-2720 AUCGTUCCAUAAUACUCUGAGAG 2001 2698-2720 AD-1136091.2 UCAGAGUAUUAUGGAACGAGU 135 2701-2721 ACUCGUUCCAUAAUACUCUGAGA 494 2699-2721 AD-1136092.2 CAGAGUAUUAUGGAACGAGCU 136 2702-2722 AGCUCGUUCCAUAAUACUCUGAG 495 2700-2722 AD-1297665.1 AGAGUAUUAUGGAACGAGCUU 1886 2703-2723 AAGCTCGUUCCAUAAUACUCUGA 2002 2701-2723 AD-1297666.1 GAGUAUUAUGGAACGAGCUUU 1887 2704-2724 AAAGCUCGUUCCAUAAUACUCUG 2003 2702-2724 AD-1297667.1 AGUAUUAUGGAACGAGCUUUU 1888 2705-2725 AAAAGCTCGUUCCAUAAUACUCU 2004 2703-2725 AD-1297668.1 GUAUUAUGGAACGAGCUUUAU 1889 2706-2726 AUAAAGCUCGUUCCAUAAUACUC 2005 2704-2726 AD-1297669.1 UAUUAUGGAACGAGCUUUAUU 1890 2707-2727 AAUAAAGCUCGUUCCAUAAUACU 2006 2705-2727 AD-1297670.1 AUUAUGGAACGAGCUUUAUUU 1891 2708-2728 AAAUAAAGCUCGUUCCAUAAUAC 2007 2706-2728 AD-1297671.1 UUAUGGAACGAGCUUUAUUCU 1892 2709-2729 AGAAUAAAGCUCGUUCCAUAAUA 2008 2707-2729 AD-1297672.1 UAUGGAACGAGCUUUAUUCCU 1893 2710-2730 AGGAAUAAAGCUCGUUCCAUAAU 2009 2708-2730 AD-1297673.1 CUCUUCCUGGCUGCUUCUAUU 1894 3869-3889 AAUAGAAGCAGCCAGGAAGAGGG 2010 3867-3889 AD-1297674.1 UCUUCCUGGCUGCUUCUAUCU 1895 3870-3890 AGAUAGAAGCAGCCAGGAAGAGG 2011 3868-3890 AD-1297675.1 CUUCCUGGCUGCUUCUAUCUU 1896 3871-3891 AAGAUAGAAGCAGCCAGGAAGAG 2012 3869-3891 AD-1136159.2 UUCCUGGCUGCUUCUAUCUUU 202 3872-3892 AAAGAUAGAAGCAGCCAGGAAGA 561 3870-3892 AD-1136160.2 UCCUGGCUGCUUCUAUCUUCU 203 3873-3893 AGAAGAUAGAAGCAGCCAGGAAG 562 3871-3893 AD-1136161.2 CCUGGCUGCUUCUAUCUUCUU 204 3874-3894 AAGAAGAUAGAAGCAGCCAGGAA 563 3872-3894 AD-1136162.2 CUGGCUGCUUCUAUCUUCUUU 205 3875-3895 AAAGAAGAUAGAAGCAGCCAGGA 564 3873-3895 AD-1136163.2 UGGCUGCUUCUAUCUUCUUUU 206 3876-3896 AAAAGAAGAUAGAAGCAGCCAGG 565 3874-3896 AD-1297676.1 GGCUGCUUCUAUCUUCUUUGU 1897 3877-3897 ACAAAGAAGAUAGAAGCAGCCAG 2013 3875-3897 AD-1136164.2 GCUGCUUCUAUCUUCUUUGCU 207 3878-3898 AGCAAAGAAGAUAGAAGCAGCCA 566 3876-3898 AD-1136165.2 CUGCUUCUAUCUUCUUUGCCU 208 3879-3899 AGGCAAAGAAGAUAGAAGCAGCC 567 3877-3899 AD-1136166.2 UGCUUCUAUCUUCUUUGCCAU 209 3880-3900 AUGGCAAAGAAGAUAGAAGCAGC 568 3878-3900 AD-1136167.2 GCUUCUAUCUUCUUUGCCAUU 210 3881-3901 AAUGGCAAAGAAGAUAGAAGCAG 569 3879-3901 AD-1136168.2 CUUCUAUCUUCUUUGCCAUCU 211 3882-3902 AGAUGGCAAAGAAGAUAGAAGCA 570 3880-3902 AD-1136169.2 UUCUAUCUUCUUUGCCAUCAU 212 3883-3903 AUGATGGCAAAGAAGAUAGAAGC 571 3881-3903 AD-1136170.2 UCUAUCUUCUUUGCCAUCAAU 213 3884-3904 AUUGAUGGCAAAGAAGAUAGAAG 572 3882-3904 AD-1136171.2 CUAUCUUCUUUGCCAUCAAAU 214 3885-3905 AUUUGATGGCAAAGAAGAUAGAA 573 3883-3905 AD-1136172.2 UAUCUUCUUUGCCAUCAAAGU 215 3886-3906 ACUUUGAUGGCAAAGAAGAUAGA 574 3884-3906 AD-1136173.2 AUCUUCUUUGCCAUCAAAGAU 216 3887-3907 AUCUTUGAUGGCAAAGAAGAUAG 575 3885-3907 AD-1297677.1 UCUUCUUUGCCAUCAAAGAUU 1898 3888-3908 AAUCUUUGAUGGCAAAGAAGAUA 2014 3886-3908 AD-1136174.2 CUUCUUUGCCAUCAAAGAUGU 217 3889-3909 ACAUCUUUGAUGGCAAAGAAGAU 576 3887-3909 AD-1297678.1 UUCUUUGCCAUCAAAGAUGCU 1899 3890-3910 AGCATCTUUGAUGGCAAAGAAGA 2015 3888-3910 AD-1297679.1 UCUUUGCCAUCAAAGAUGCCU 1900 3891-3911 AGGCAUCUUUGAUGGCAAAGAAG 2016 3889-3911 AD-1297680.1 CUUUGCCAUCAAAGAUGCCAU 1901 3892-3912 AUGGCATCUUUGAUGGCAAAGAA 2017 3890-3912 AD-1297681.1 UUUGCCAUCAAAGAUGCCAUU 1902 3893-3913 AAUGGCAUCUUUGAUGGCAAAGA 2018 3891-3913 AD-1297682.1 UUGCCAUCAAAGAUGCCAUCU 1903 3894-3914 AGAUGGCAUCUUUGAUGGCAAAG 2019 3892-3914 AD-1297683.1 UGCCAUCAAAGAUGCCAUCCU 1904 3895-3915 AGGATGGCAUCUUUGAUGGCAAA 2020 3893-3915 AD-1297684.1 GCCAUCAAAGAUGCCAUCCGU 1905 3896-3916 ACGGAUGGCAUCUUUGAUGGCAA 2021 3894-3916 AD-1297685.1 AAACCAAUGAACAGCAAAGCU 1906 4512-4532 AGCUTUGCUGUUCAUUGGUUUGA 2022 4510-4532 AD-1297686.1 AACCAAUGAACAGCAAAGCAU 1907 4513-4533 AUGCTUTGCUGUUCAUUGGUUUG 2023 4511-4533 AD-1297687.1 ACCAAUGAACAGCAAAGCAUU 1908 4514-4534 AAUGCUTUGCUGUUCAUUGGUUU 2024 4512-4534 AD-1297688.1 CCAAUGAACAGCAAAGCAUAU 1909 4515-4535 AUAUGCTUUGCUGUUCAUUGGUU 2025 4513-4535 AD-1297689.1 CAAUGAACAGCAAAGCAUAAU 1910 4516-4536 AUUATGCUUUGCUGUUCAUUGGU 2026 4514-4536 AD-1297690.1 AAUGAACAGCAAAGCAUAACU 1911 4517-4537 AGUUAUGCUUUGCUGUUCAUUGG 2027 4515-4537 AD-1297691.1 AUGAACAGCAAAGCAUAACCU 1912 4518-4538 AGGUTATGCUUUGCUGUUCAUUG 2028 4516-4538 AD-1297692.1 UGAACAGCAAAGCAUAACCUU 1913 4519-4539 AAGGUUAUGCUUUGCUGUUCAUU 2029 4517-4539 AD-1136221.2 GAACAGCAAAGCAUAACCUUU 264 4520-4540 AAAGGUUAUGCUUUGCUGUUCAU 623 4518-4540 AD-1136222.2 AACAGCAAAGCAUAACCUUGU 265 4521-4541 ACAAGGTUAUGCUUUGCUGUUCA 624 4519-4541 AD-1297693.1 ACAGCAAAGCAUAACCUUGAU 1914 4522-4542 AUCAAGGUUAUGCUUUGCUGUUC 2030 4520-4542 AD-1297694.1 CAGCAAAGCAUAACCUUGAAU 1915 4523-4543 AUUCAAGGUUAUGCUUUGCUGUU 2031 4521-4543 AD-1136223.2 AGCAAAGCAUAACCUUGAAUU 266 4524-4544 AAUUCAAGGUUAUGCUUUGCUGU 625 4522-4544 AD-1136224.2 GCAAAGCAUAACCUUGAAUCU 267 4525-4545 AGAUTCAAGGUUAUGCUUUGCUG 626 4523-4545 AD-1136225.2 CAAAGCAUAACCUUGAAUCUU 268 4526-4546 AAGATUCAAGGUUAUGCUUUGCU 627 4524-4546 AD-1297695.1 AAAGCAUAACCUUGAAUCUAU 1916 4527-4547 AUAGAUTCAAGGUUAUGCUUUGC 2032 4525-4547 AD-1297696.1 AAGCAUAACCUUGAAUCUAUU 1917 4528-4548 AAUAGAUUCAAGGUUAUGCUUUG 2033 4526-4548 AD-1297697.1 AGCAUAACCUUGAAUCUAUAU 1918 4529-4549 AUAUAGAUUCAAGGUUAUGCUUU 2034 4527-4549 AD-1136226.2 GCAUAACCUUGAAUCUAUACU 269 4530-4550 AGUAUAGAUUCAAGGUUAUGCUU 628 4528-4550 AD-1136227.2 CAUAACCUUGAAUCUAUACUU 270 4531-4551 AAGUAUAGAUUCAAGGUUAUGCU 629 4529-4551 AD-1136228.2 AUAACCUUGAAUCUAUACUCU 271 4532-4552 AGAGUAUAGAUUCAAGGUUAUGC 630 4530-4552 AD-1136229.2 UAACCUUGAAUCUAUACUCAU 272 4533-4553 AUGAGUAUAGAUUCAAGGUUAUG 631 4531-4553 AD-1136230.2 AACCUUGAAUCUAUACUCAAU 273 4534-4554 AUUGAGTAUAGAUUCAAGGUUAU 632 4532-4554 AD-1136231.2 ACCUUGAAUCUAUACUCAAAU 274 4535-4555 AUUUGAGUAUAGAUUCAAGGUUA 633 4533-4555 AD-1136232.2 CCUUGAAUCUAUACUCAAAUU 275 4536-4556 AAUUTGAGUAUAGAUUCAAGGUU 634 4534-4556 AD-1297698.1 CUUGAAUCUAUACUCAAAUUU 1919 4537-4557 AAAUTUGAGUAUAGAUUCAAGGU 2035 4535-4557 AD-1297699.1 UUGAAUCUAUACUCAAAUUUU 1920 4538-4558 AAAAUUUGAGUAUAGAUUCAAGG 2036 4536-4558 AD-1297700.1 GAAUCUAUACUCAAAUUUUGU 1921 4540-4560 ACAAAAUUUGAGUAUAGAUUCAA 2037 4538-4560 AD-1136233.2 AAUCUAUACUCAAAUUUUGCU 276 4541-4561 AGCAAAAUUUGAGUAUAGAUUCA 635 4539-4561 AD-1136234.2 AUCUAUACUCAAAUUUUGCAU 277 4542-4562 AUGCAAAAUUUGAGUAUAGAUUC 636 4540-4562 AD-1297701.1 UCUAUACUCAAAUUUUGCAAU 1922 4543-4563 AUUGCAAAAUUUGAGUAUAGAUU 2038 4541-4563 AD-1297702.1 CUAUACUCAAAUUUUGCAAUU 1923 4544-4564 AAUUGCAAAAUUUGAGUAUAGAU 2039 4542-4564 AD-1297703.1 UAUACUCAAAUUUUGCAAUGU 1924 4545-4565 ACAUTGCAAAAUUUGAGUAUAGA 2040 4543-4565 AD-1297704.1 AUACUCAAAUUUUGCAAUGAU 1925 4546-4566 AUCATUGCAAAAUUUGAGUAUAG 2041 4544-4566 AD-1297705.1 UACUCAAAUUUUGCAAUGAGU 1926 4547-4567 ACUCAUUGCAAAAUUUGAGUAUA 2042 4545-4567 AD-1297706.1 ACUCAAAUUUUGCAAUGAGGU 1927 4548-4568 ACCUCAUUGCAAAAUUUGAGUAU 2043 4546-4568 AD-1297707.1 CUCAAAUUUUGCAAUGAGGCU 1928 4549-4569 AGCCTCAUUGCAAAAUUUGAGUA 2044 4547-4569 AD-1297708.1 UCAAAUUUUGCAAUGAGGCAU 1929 4550-4570 AUGCCUCAUUGCAAAAUUUGAGU 2045 4548-4570 AD-1297709.1 CAAAUUUUGCAAUGAGGCAGU 1930 4551-4571 ACUGCCTCAUUGCAAAAUUUGAG 2046 4549-4571 AD-1297710.1 AAAUUUUGCAAUGAGGCAGUU 1931 4552-4572 AACUGCCUCAUUGCAAAAUUUGA 2047 4550-4572 AD-1297711.1 AAUUUUGCAAUGAGGCAGUGU 1932 4553-4573 ACACTGCCUCAUUGCAAAAUUUG 2048 4551-4573 AD-1297712.1 AUUUUGCAAUGAGGCAGUGGU 1933 4554-4574 ACCACUGCCUCAUUGCAAAAUUU 2049 4552-4574 AD-1395794.1 CUCUCCAAGUAUGAUCGUCUU 40 239-259 AAGACGAUCAUACUUGGAGAGCG 2050 237-259 AD-1395797.1 AGGAUCUCUCUCAGAGUAUUU 126 2691-2711 AAAUACTCUGAGAGAGAUCCUGG 485 2689-2711 AD-1395803.1 CAGUGAUGCUCUCCAAGUAUU 1819 231-251 AAUACUTGGAGAGCAUCACUGUG 2051 229-251 AD-1395805.1 CCAAGUAUGAUCGUCUGCAGU 1826 243-263 ACUGCAGACGATCAUACUUGGAG 2052 241-263 AD-1395807.1 GUUUGUUUCAUUUCCGCUGAU 1853 1973-1993 ATCAGCGGAAATGAAACAAACGG 2053 1971-1993 AD-1395811.1 AACACCCAGGAUCUCUCUCAU 1878 2684-2704 ATGAGAGAGAUCCUGGGUGUUCC 2054 2682-2704 AD-1395816.1 GGGUUUGUUUGUUUCAUUUCU 96 1967-1987 AGAAAUGAAACAAACAAACCCUG 455 1965-1987 AD-1395823.1 GAGUAUUUCUCAGCAUUCAAU 83 1322-1342 ATUGAATGCUGAGAAAUACUCCC 2055 1320-1342

TABLE 7 Modified Sense and Antisense Strand Sequences of Xanthine Dehydrogenase dsRNA Agents SEQ SEQ mRNA SEQ Duplex ID ID Target ID Name Sense Sequence 5′ to 3′ NO: Antisense Sequence 5′ to 3′ NO: Sequence NO: AD- gscsacagUfgAfUfGfcucuccaaguL96 752 asCfsuudGg(Agn)gagcauCfaCfugugc 1111 UUGCACAGUGAU 1470 1135990.2 sasa GCUCUCCAAGU AD- csascaguGfaUfGfCfucuccaaguuL96 753 asAfscudTg(G2p)agagcaUfcAfcugug 1112 UGCACAGUGAUG 1471 1135991.2 scsa CUCUCCAAGUA AD- ascsagugAfuGfCfUfcuccaaguauL96 2056 asUfsacdTu(G2p)gagagcAfuCfacugu 2180 GCACAGUGAUGC 2304 1297597.1 sgsc UCUCCAAGUAU AD- csasgugaUfgCfUfCfuccaaguauuL96 2057 asAfsuacUfuggagagCfaUfcacugsusg 2181 CACAGUGAUGCU 2305 1297598.1 CUCCAAGUAUG AD- asgsugauGfcUfCfUfccaaguauguL96 2058 asCfsauaCfuuggagaGfcAfucacusgsu 2182 ACAGUGAUGCUC 2306 1297599.1 UCCAAGUAUGA AD- gsusgaugCfuCfUfCfcaaguaugauL96 2059 asUfscauAfcuuggagAfgCfaucacsusg 2183 CAGUGAUGCUCU 2307 1297600.1 CCAAGUAUGAU AD- usgsaugcUfcUfCfCfaaguaugauuL96 2060 asAfsucaUfacuuggaGfaGfcaucascsu 2184 AGUGAUGCUCUC 2308 1297601.1 CAAGUAUGAUC AD- gsasugcuCfuCfCfAfaguaugaucuL96 754 asGfsaucAfuacuuggAfgAfgcaucsasc 1113 GUGAUGCUCUCC 1472 1135992.2 AAGUAUGAUCG AD- asusgcucUfcCfAfAfguaugaucguL96 755 asCfsgauCfauacuugGfaGfagcauscsa 1114 UGAUGCUCUCCA 1473 1135993.2 AGUAUGAUCGU AD- usgscucuCfcAfAfGfuaugaucguuL96 756 asAfscgaUfcauacuuGfgAfgagcasusc 1115 GAUGCUCUCCAA 1474 1135994.2 GUAUGAUCGUC AD- gscsucucCfaAfGfUfaugaucgucuL96 757 asGfsacgAfucauacuUfgGfagagcsasu 1116 AUGCUCUCCAAG 1475 1135995.2 UAUGAUCGUCU AD- csuscuccAfaGfUfAfugaucgucuuL96 758 asAfsgacGfaucauacUfuGfgagagscsa 1117 UGCUCUCCAAGU 1476 1135996.2 AUGAUCGUCUG AD- uscsuccaAfgUfAfUfgaucgucuguL96 2061 asCfsagaCfgaucauaCfuUfggagasgsc 2185 GCUCUCCAAGUA 2309 1297602.1 UGAUCGUCUGC AD- csusccaaGfuAfUfGfaucgucugcuL96 2062 asGfscadGa(C2p)gaucauAfcUfuggag 2186 CUCUCCAAGUAU 2310 1297603.1 sasg GAUCGUCUGCA AD- uscscaagUfaUfGfAfucgucugcauL96 2063 asUfsgcdAg(Agn)cgaucaUfaCfuugga 2187 UCUCCAAGUAUG 2311 1297604.1 sgsa AUCGUCUGCAG AD- cscsaaguAfuGfAfUfcgucugcaguL96 2064 asCfsugcAfgacgaucAfuAfcuuggsasg 2188 CUCCAAGUAUGA 2312 1297605.1 UCGUCUGCAGA AD- csasaguaUfgAfUfCfgucugcagauL96 2065 asUfscudGc(Agn)gacgauCfaUfacuug 2189 UCCAAGUAUGAU 2313 1297606.1 sgsa CGUCUGCAGAA AD- asasguauGfaUfCfGfucugcagaauL96 2066 asUfsucdTg(C2p)agacgaUfcAfuacuu 2190 CCAAGUAUGAUC 2314 1297607.1 sgsg GUCUGCAGAAC AD- asgsuaugAfuCfGfUfcugcagaacuL96 2067 asGfsuudCu(G2p)cagacgAfuCfauacu 2191 CAAGUAUGAUCG 2315 1297608.1 susg UCUGCAGAACA AD- gsusaugaUfcGfUfCfugcagaacauL96 2068 asUfsgudTc(Tgn)gcagacGfaUfcauac 2192 AAGUAUGAUCGU 2316 1297609.1 susu CUGCAGAACAA AD- usasugauCfgUfCfUfgcagaacaauL96 2069 asUfsugdTu(C2p)ugcagaCfgAfucaua 2193 AGUAUGAUCGUC 2317 1297610.1 scsu UGCAGAACAAG AD- asusgaucGfuCfUfGfcagaacaaguL96 2070 asCfsuugUfucugcagAfcGfaucausasc 2194 GUAUGAUCGUCU 2318 1297611.1 GCAGAACAAGA AD- gsgsgaguAfuUfUfCfucagcauucuL96 799 asGfsaadTg(C2p)ugagaaAfuAfcuccc 1158 GGGGGAGUAUUU 1517 1136037.2 scsc CUCAGCAUUCA AD- gsgsaguaUfuUfCfUfcagcauucauL96 800 asUfsgadAu(G2p)cugagaAfaUfacucc 1159 GGGGAGUAUUUC 1518 1136038.2 scsc UCAGCAUUCAA AD- gsasguauUfuCfUfCfagcauucaauL96 801 asUfsugaAfugcugagAfaAfuacucscsc 1160 GGGAGUAUUUCU 1519 1136039.2 CAGCAUUCAAG AD- asgsuauuUfcUfCfAfgcauucaaguL96 802 asCfsuugAfaugcugaGfaAfauacuscsc 1161 GGAGUAUUUCUC 1520 1136040.2 AGCAUUCAAGC AD- gsusauuuCfuCfAfGfcauucaagcuL96 803 asGfscudTg(Agn)augcugAfgAfaauac 1162 GAGUAUUUCUCA 1521 1136041.2 susc GCAUUCAAGCA AD- usasuuucUfcAfGfCfauucaagcauL96 2071 asUfsgcdTu(G2p)aaugcuGfaGfaaaua 2195 AGUAUUUCUCAG 2319 1297612.1 scsu CAUUCAAGCAG AD- asusuucuCfaGfCfAfuucaagcaguL96 2072 asCfsugcUfugaaugcUfgAfgaaausasc 2196 GUAUUUCUCAGC 2320 1297613.1 AUUCAAGCAGG AD- ususucucAfgCfAfUfucaagcagguL96 2073 asCfscudGc(Tgn)ugaaugCfuGfagaaa 2197 UAUUUCUCAGCA 2321 1297614.1 susa UUCAAGCAGGC AD- ususcucaGfcAfUfUfcaagcaggcuL96 2074 asGfsccdTg(C2p)uugaauGfcUfgagaa 2198 AUUUCUCAGCAU 2322 1297615.1 sasu UCAAGCAGGCC AD- uscsucagCfaUfUfCfaagcaggccuL96 2075 asGfsgcdCu(G2p)cuugaaUfgCfugaga 2199 UUUCUCAGCAUU 2323 1297616.1 sasa CAAGCAGGCCU AD- csuscagcAfuUfCfAfagcaggccuuL96 2076 asAfsggdCc(Tgn)gcuugaAfuGfcugag 2200 UUCUCAGCAUUC 2324 1297617.1 sasa AAGCAGGCCUC AD- uscsagcaUfuCfAfAfgcaggccucuL96 2077 asGfsagdGc(C2p)ugcuugAfaUfgcuga 2201 UCUCAGCAUUCA 2325 1297618.1 sgsa AGCAGGCCUCC AD- csasgcauUfcAfAfGfcaggccuccuL96 2078 asGfsgadGg(C2p)cugcuuGfaAfugcug 2202 CUCAGCAUUCAA 2326 1297619.1 sasg GCAGGCCUCCC AD- asasgaagGfuUfCfCfaggguuuguuL96 2079 asAfscaaAfcccuggaAfcCfuucuusasg 2203 CUAAGAAGGUUC 2327 1297620.1 CAGGGUUUGUU AD- asgsaaggUfuCfCfAfggguuuguuuL96 2080 asAfsacaAfacccuggAfaCfcuucususa 2204 UAAGAAGGUUCC 2328 1297621.1 AGGGUUUGUUU AD- gsasagguUfcCfAfGfgguuuguuuuL96 2081 asAfsaacAfaacccugGfaAfccuucsusu 2205 AAGAAGGUUCCA 2329 1297622.1 GGGUUUGUUUG AD- asasgguuCfcAfGfGfguuuguuuguL96 2082 asCfsaaaCfaaacccuGfgAfaccuuscsu 2206 AGAAGGUUCCAG 2330 1297623.1 GGUUUGUUUGU AD- asgsguucCfaGfGfGfuuuguuuguuL96 2083 asAfscaaAfcaaacccUfgGfaaccususc 2207 GAAGGUUCCAGG 2331 1297624.1 GUUUGUUUGUU AD- gsgsuuccAfgGfGfUfuuguuuguuuL96 2084 asAfsacaAfacaaaccCfuGfgaaccsusu 2208 AAGGUUCCAGGG 2332 1297625.1 UUUGUUUGUUU AD- gsusuccaGfgGfUfUfuguuuguuuuL96 2085 asAfsaacAfaacaaacCfcUfggaacscsu 2209 AGGUUCCAGGGU 2333 1297626.1 UUGUUUGUUUC AD- ususccagGfgUfUfUfguuuguuucuL96 812 asGfsaaaCfaaacaaaCfcCfuggaascsc 1171 GGUUCCAGGGUU 1530 1136050.2 UGUUUGUUUCA AD- uscscaggGfuUfUfGfuuuguuucauL96 2086 asUfsgaaAfcaaacaaAfcCfcuggasasc 2210 GUUCCAGGGUUU 2334 1297627.1 GUUUGUUUCAU AD- cscsagggUfuUfGfUfuuguuucauuL96 2087 asAfsugaAfacaaacaAfaCfccuggsasa 2211 UUCCAGGGUUUG 2335 1297628.1 UUUGUUUCAUU AD- csasggguUfuGfUfUfuguuucauuuL96 813 asAfsaugAfaacaaacAfaAfcccugsgsa 1172 UCCAGGGUUUGU 1531 1136051.2 UUGUUUCAUUU AD- gsgsguuuGfuUfUfGfuuucauuucuL96 814 asGfsaaaUfgaaacaaAfcAfaacccsusg 1173 CAGGGUUUGUUU 1532 1136052.2 GUUUCAUUUCC AD- gsgsuuugUfuUfGfUfuucauuuccuL96 815 asGfsgaaAfugaaacaAfaCfaaaccscsu 1174 AGGGUUUGUUUG 1533 1136053.2 UUUCAUUUCCG AD- gsusuuguUfuGfUfUfucauuuccguL96 2088 asCfsggaAfaugaaacAfaAfcaaacscsc 2212 GGGUUUGUUUGU 2336 1297629.1 UUCAUUUCCGC AD- ususuguuUfgUfUfUfcauuuccgcuL96 2089 asGfscggAfaaugaaaCfaAfacaaascsc 2213 GGUUUGUUUGUU 2337 1297630.1 UCAUUUCCGCU AD- ususguuuGfuUfUfCfauuuccgcuuL96 816 asAfsgcgGfaaaugaaAfcAfaacaasasc 1175 GUUUGUUUGUUU 1534 1136054.2 CAUUUCCGCUG AD- usgsuuugUfuUfCfAfuuuccgcuguL96 2090 asCfsagcGfgaaaugaAfaCfaaacasasa 2214 UUUGUUUGUUUC 2338 1297631.1 AUUUCCGCUGA AD- gsusuuguUfuCfAfUfuuccgcugauL96 2091 asUfscadGc(G2p)gaaaugAfaAfcaaac 2215 UUGUUUGUUUCA 2339 1297632.1 sasa UUUCCGCUGAU AD- ususuguuUfcAfUfUfuccgcugauuL96 2092 asAfsucdAg(C2p)ggaaauGfaAfacaaa 2216 UGUUUGUUUCAU 2340 1297633.1 scsa UUCCGCUGAUG AD- ususguuuCfaUfUfUfccgcugauguL96 2093 asCfsaudCa(G2p)cggaaaUfgAfaacaa 2217 GUUUGUUUCAUU 2341 1297634.1 sasc UCCGCUGAUGA AD- usgsuuucAfuUfUfCfcgcugaugauL96 2094 asUfscadTc(Agn)gcggaaAfuGfaaaca 2218 UUUGUUUCAUUU 2342 1297635.1 sasa CCGCUGAUGAU AD- gsusuucaUfuUfCfCfgcugaugauuL96 2095 asAfsucdAu(C2p)agcggaAfaUfgaaac 2219 UUGUUUCAUUUC 2343 1297636.1 sasa CGCUGAUGAUG AD- ususucauUfuCfCfGfcugaugauguL96 2096 asCfsaucAfucagcggAfaAfugaaascsa 2220 UGUUUCAUUUCC 2344 1297637.1 GCUGAUGAUGU AD- gsgsagauGfgAfGfCfucuuuguguuL96 2097 asAfscacAfaagagcuCfcAfucuccscsc 2221 GGGGAGAUGGAG 2345 1297638.1 CUCUUUGUGUC AD- gsasgaugGfaGfCfUfcuuugugucuL96 2098 asGfsacaCfaaagagcUfcCfaucucscsc 2222 GGGAGAUGGAGC 2346 1297639.1 UCUUUGUGUCU AD- asgsauggAfgCfUfCfuuugugucuuL96 2099 asAfsgadCa(C2p)aaagagCfuCfcaucu 2223 GGAGAUGGAGCU 2347 1297640.1 scsc CUUUGUGUCUA AD- gsasuggaGfcUfCfUfuugugucuauL96 2100 asUfsagdAc(Agn)caaagaGfcUfccauc 2224 GAGAUGGAGCUC 2348 1297641.1 susc UUUGUGUCUAC AD- asusggagCfuCfUfUfugugucuacuL96 2101 asGfsuadGa(C2p)acaaagAfgCfuccau 2225 AGAUGGAGCUCU 2349 1297642.1 scsu UUGUGUCUACA AD- usgsgagcUfcUfUfUfgugucuacauL96 2102 asUfsgudAg(Agn)cacaaaGfaGfcucca 2226 GAUGGAGCUCUU 2350 1297643.1 susc UGUGUCUACAC AD- gsgsagcuCfuUfUfGfugucuacacuL96 2103 asGfsugdTa(G2p)acacaaAfgAfgcucc 2227 AUGGAGCUCUUU 2351 1297644.1 sasu GUGUCUACACA AD- gsasgcucUfuUfGfUfgucuacacauL96 2104 asUfsgudGu(Agn)gacacaAfaGfagcuc 2228 UGGAGCUCUUUG 2352 1297645.1 scsa UGUCUACACAG AD- asgscucuUfuGfUfGfucuacacaguL96 835 asCfsugdTg(Tgn)agacacAfaAfgagcu 1194 GGAGCUCUUUGU 1553 1136073.2 scsc GUCUACACAGA AD- gscsucuuUfgUfGfUfcuacacagauL96 836 asUfscudGu(G2p)uagacaCfaAfagagc 1195 GAGCUCUUUGUG 1554 1136074.2 susc UCUACACAGAA AD- csuscuuuGfuGfUfCfuacacagaauL96 837 asUfsucdTg(Tgn)guagacAfcAfaagag 1196 AGCUCUUUGUGU 1555 1136075.2 scsu CUACACAGAAC AD- uscsuuugUfgUfCfUfacacagaacuL96 838 asGfsuudCu(G2p)uguagaCfaCfaaaga 1197 GCUCUUUGUGUC 1556 1136076.2 sgsc UACACAGAACA AD- csusuuguGfuCfUfAfcacagaacauL96 839 asUfsgudTc(Tgn)guguagAfcAfcaaag 1198 CUCUUUGUGUCU 1557 1136077.2 sasg ACACAGAACAC AD- ususugugUfcUfAfCfacagaacacuL96 840 asGfsugdTu(C2p)uguguaGfaCfacaaa 1199 UCUUUGUGUCUA 1558 1136078.2 sgsa CACAGAACACC AD- ususguguCfuAfCfAfcagaacaccuL96 2105 asGfsgudGu(Tgn)cuguguAfgAfcacaa 2229 CUUUGUGUCUAC 2353 1297646.1 sasg ACAGAACACCA AD- usgsugucUfaCfAfCfagaacaccauL96 2106 asUfsggdTg(Tgn)ucugugUfaGfacaca 2230 UUUGUGUCUACA 2354 1297647.1 sasa CAGAACACCAU AD- gsusgucuAfcAfCfAfgaacaccauuL96 2107 asAfsugdGu(G2p)uucuguGfuAfgacac 2231 UUGUGUCUACAC 2355 1297648.1 sasa AGAACACCAUG AD- usgsucuaCfaCfAfGfaacaccauguL96 2108 asCfsaudGg(Tgn)guucugUfgUfagaca 2232 UGUGUCUACACA 2356 1297649.1 scsa GAACACCAUGA AD- gsuscuacAfcAfGfAfacaccaugauL96 2109 asUfscadTg(G2p)uguucuGfuGfuagac 2233 GUGUCUACACAG 2357 1297650.1 sasc AACACCAUGAA AD- uscsuacaCfaGfAfAfcaccaugaauL96 2110 asUfsucdAu(G2p)guguucUfgUfguaga 2234 UGUCUACACAGA 2358 1297651.1 scsa ACACCAUGAAG AD- csusacacAfgAfAfCfaccaugaaguL96 2111 asCfsuucAfugguguuCfuGfuguagsasc 2235 GUCUACACAGAA 2359 1297652.1 CACCAUGAAGA AD- usascacaGfaAfCfAfccaugaagauL96 2112 asUfscudTc(Agn)ugguguUfcUfgugua 2236 UCUACACAGAAC 2360 1297653.1 sgsa ACCAUGAAGAC AD- gsgsgaacAfcCfCfAfggaucucucuL96 2113 asGfsagdAg(Agn)uccuggGfuGfuuccc 2237 UGGGGAACACCC 2361 1297654.1 scsa AGGAUCUCUCU AD- gsgsaacaCfcCfAfGfgaucucucuuL96 2114 asAfsgadGa(G2p)auccugGfgUfguucc 2238 GGGGAACACCCA 2362 1297655.1 scsc GGAUCUCUCUC AD- gsasacacCfcAfGfGfaucucucucuL96 2115 asGfsagdAg(Agn)gauccuGfgGfuguuc 2239 GGGAACACCCAG 2363 1297656.1 scsc GAUCUCUCUCA AD- asascaccCfaGfGfAfucucucucauL96 2116 asUfsgadGa(G2p)agauccUfgGfguguu 2240 GGAACACCCAGG 2364 1297657.1 scsc AUCUCUCUCAG AD- ascsacccAfgGfAfUfcucucucaguL96 2117 asCfsugaGfagagaucCfuGfggugususc 2241 GAACACCCAGGA 2365 1297658.1 UCUCUCUCAGA AD- csascccaGfgAfUfCfucucucagauL96 2118 asUfscudGa(G2p)agagauCfcUfgggug 2242 AACACCCAGGAU 2366 1297659.1 susu CUCUCUCAGAG AD- ascsccagGfaUfCfUfcucucagaguL96 2119 asCfsucdTg(Agn)gagagaUfcCfugggu 2243 ACACCCAGGAUC 2367 1297660.1 sgsu UCUCUCAGAGU AD- cscscaggAfuCfUfCfucucagaguuL96 2120 asAfscudCu(G2p)agagagAfuCfcuggg 2244 CACCCAGGAUCU 2368 1297661.1 susg CUCUCAGAGUA AD- cscsaggaUfcUfCfUfcucagaguauL96 2121 asUfsacdTc(Tgn)gagagaGfaUfccugg 2245 ACCCAGGAUCUC 2369 1297662.1 sgsu UCUCAGAGUAU AD- csasggauCfuCfUfCfucagaguauuL96 2122 asAfsuadCu(C2p)ugagagAfgAfuccug 2246 CCCAGGAUCUCU 2370 1297663.1 sgsg CUCAGAGUAUU AD- asgsgaucUfcUfCfUfcagaguauuuL96 844 asAfsaudAc(Tgn)cugagaGfaGfauccu 1203 CCAGGAUCUCUC 1562 1136082.2 sgsg UCAGAGUAUUA AD- gsgsaucuCfuCfUfCfagaguauuauL96 845 asUfsaadTa(C2p)ucugagAfgAfgaucc 1204 CAGGAUCUCUCU 1563 1136083.2 susg CAGAGUAUUAU AD- gsasucucUfcUfCfAfgaguauuauuL96 846 asAfsuaaUfacucugaGfaGfagaucscsu 1205 AGGAUCUCUCUC 1564 1136084.2 AGAGUAUUAUG AD- asuscucuCfuCfAfGfaguauuauguL96 847 asCfsauaAfuacucugAfgAfgagauscsc 1206 GGAUCUCUCUCA 1565 1136085.2 GAGUAUUAUGG AD- uscsucucUfcAfGfAfguauuaugguL96 848 asCfscauAfauacucuGfaGfagagasusc 1207 GAUCUCUCUCAG 1566 1136086.2 AGUAUUAUGGA AD- csuscucuCfaGfAfGfuauuauggauL96 849 asUfsccaUfaauacucUfgAfgagagsasu 1208 AUCUCUCUCAGA 1567 1136087.2 GUAUUAUGGAA AD- uscsucucAfgAfGfUfauuauggaauL96 850 asUfsuccAfuaauacuCfuGfagagasgsa 1209 UCUCUCUCAGAG 1568 1136088.2 UAUUAUGGAAC AD- csuscucaGfaGfUfAfuuauggaacuL96 851 asGfsuudCc(Agn)uaauacUfcUfgagag 1210 CUCUCUCAGAGU 1569 1136089.2 sasg AUUAUGGAACG AD- uscsucagAfgUfAfUfuauggaacguL96 852 asCfsguuCfcauaauaCfuCfugagasgsa 1211 UCUCUCAGAGUA 1570 1136090.2 UUAUGGAACGA AD- csuscagaGfuAfUfUfauggaacgauL96 2123 asUfscgdTu(C2p)cauaauAfcUfcugag 2247 CUCUCAGAGUAU 2371 1297664.1 sasg UAUGGAACGAG AD- uscsagagUfaUfUfAfuggaacgaguL96 853 asCfsucgUfuccauaaUfaCfucugasgsa 1212 UCUCAGAGUAUU 1571 1136091.2 AUGGAACGAGC AD- csasgaguAfuUfAfUfggaacgagcuL96 854 asGfscucGfuuccauaAfuAfcucugsasg 1213 CUCAGAGUAUUA 1572 1136092.2 UGGAACGAGCU AD- asgsaguaUfuAfUfGfgaacgagcuuL96 2124 asAfsgcdTc(G2p)uuccauAfaUfacucu 2248 UCAGAGUAUUAU 2372 1297665.1 sgsa GGAACGAGCUU AD- gsasguauUfaUfGfGfaacgagcuuuL96 2125 asAfsagdCu(C2p)guuccaUfaAfuacuc 2249 CAGAGUAUUAUG 2373 1297666.1 susg GAACGAGCUUU AD- asgsuauuAfuGfGfAfacgagcuuuuL96 2126 asAfsaadGc(Tgn)cguuccAfuAfauacu 2250 AGAGUAUUAUGG 2374 1297667.1 scsu AACGAGCUUUA AD- gsusauuaUfgGfAfAfcgagcuuuauL96 2127 asUfsaadAg(C2p)ucguucCfaUfaauac 2251 GAGUAUUAUGGA 2375 1297668.1 susc ACGAGCUUUAU AD- usasuuauGfgAfAfCfgagcuuuauuL96 2128 asAfsuadAa(G2p)cucguuCfcAfuaaua 2252 AGUAUUAUGGAA 2376 1297669.1 scsu CGAGCUUUAUU AD- asusuaugGfaAfCfGfagcuuuauuuL96 2129 asAfsauaAfagcucguUfcCfauaausasc 2253 GUAUUAUGGAAC 2377 1297670.1 GAGCUUUAUUC AD- ususauggAfaCfGfAfgcuuuauucuL96 2130 asGfsaauAfaagcucgUfuCfcauaasusa 2254 UAUUAUGGAACG 2378 1297671.1 CAGCUUUAUUC AD- usasuggaAfcGfAfGfcuuuauuccuL96 2131 asGfsgaaUfaaagcucGfuUfccauasasu 2255 AUUAUGGAACGA 2379 1297672.1 GCUUUAUUCCA AD- csuscuucCfuGfGfCfugcuucuauuL96 2132 asAfsuagAfagcagccAfgGfaagagsgsg 2256 CCCUCUUCCUGG 2380 1297673.1 CUGCUUCUAUC AD- uscsuuccUfgGfCfUfgcuucuaucuL96 2133 asGfsaudAg(Agn)agcagcCfaGfgaaga 2257 CCUCUUCCUGGC 2381 1297674.1 sgsg UGCUUCUAUCU AD- csusuccuGfgCfUfGfcuucuaucuuL96 2134 asAfsgauAfgaagcagCfcAfggaagsasg 2258 CUCUUCCUGGCU 2382 1297675.1 GCUUCUAUCUU AD- ususccugGfcUfGfCfuucuaucuuuL96 920 asAfsagaUfagaagcaGfcCfaggaasgsa 1279 UCUUCCUGGCUG 1638 1136159.2 CUUCUAUCUUC AD- uscscuggCfuGfCfUfucuaucuucuL96 921 asGfsaagAfuagaagcAfgCfcaggasasg 1280 CUUCCUGGCUGC 1639 1136160.2 UUCUAUCUUCU AD- cscsuggcUfgCfUfUfcuaucuucuuL96 922 asAfsgaaGfauagaagCfaGfccaggsasa 1281 UUCCUGGCUGCU 1640 1136161.2 UCUAUCUUCUU AD- csusggcuGfcUfUfCfuaucuucuuuL96 923 asAfsagaAfgauagaaGfcAfgccagsgsa 1282 UCCUGGCUGCUU 1641 1136162.2 CUAUCUUCUUU AD- usgsgcugCfuUfCfUfaucuucuuuuL96 924 asAfsaagAfagauagaAfgCfagccasgsg 1283 CCUGGCUGCUUC 1642 1136163.2 UAUCUUCUUUG AD- gsgscugcUfuCfUfAfucuucuuuguL96 2135 asCfsaaaGfaagauagAfaGfcagccsasg 2259 CUGGCUGCUUCU 2383 1297676.1 AUCUUCUUUGC AD- gscsugcuUfcUfAfUfcuucuuugcuL96 925 asGfscaaAfgaagauaGfaAfgcagcscsa 1284 UGGCUGCUUCUA 1643 1136164.2 UCUUCUUUGCC AD- csusgcuuCfuAfUfCfuucuuugccuL96 926 asGfsgcaAfagaagauAfgAfagcagscsc 1285 GGCUGCUUCUAU 1644 1136165.2 CUUCUUUGCCA AD- usgscuucUfaUfCfUfucuuugccauL96 927 asUfsggdCa(Agn)agaagaUfaGfaagca 1286 GCUGCUUCUAUC 1645 1136166.2 sgsc UUCUUUGCCAU AD- gscsuucuAfuCfUfUfcuuugccauuL96 928 asAfsugdGc(Agn)aagaagAfuAfgaagc 1287 CUGCUUCUAUCU 1646 1136167.2 sasg UCUUUGCCAUC AD- csusucuaUfcUfUfCfuuugccaucuL96 929 asGfsaudGg(C2p)aaagaaGfaUfagaag 1288 UGCUUCUAUCUU 1647 1136168.2 scsa CUUUGCCAUCA AD- ususcuauCfuUfCfUfuugccaucauL96 930 asUfsgadTg(G2p)caaagaAfgAfuagaa 1289 GCUUCUAUCUUC 1648 1136169.2 sgsc UUUGCCAUCAA AD- uscsuaucUfuCfUfUfugccaucaauL96 931 asUfsugdAu(G2p)gcaaagAfaGfauaga 1290 CUUCUAUCUUCU 1649 1136170.2 sasg UUGCCAUCAAA AD- csusaucuUfcUfUfUfgccaucaaauL96 932 asUfsuudGa(Tgn)ggcaaaGfaAfgauag 1291 UUCUAUCUUCUU 1650 1136171.2 sasa UGCCAUCAAAG AD- usasucuuCfuUfUfGfccaucaaaguL96 933 asCfsuuuGfauggcaaAfgAfagauasgsa 1292 UCUAUCUUCUUU 1651 1136172.2 GCCAUCAAAGA AD- asuscuucUfuUfGfCfcaucaaagauL96 934 asUfscudTu(G2p)auggcaAfaGfaagau 1293 CUAUCUUCUUUG 1652 1136173.2 sasg UCCAUCAAAGA AD- uscsuucuUfuGfCfCfaucaaagauuL96 2136 asAfsucuUfugauggcAfaAfgaagasusa 2260 UAUCUUCUUUGC 2384 1297677.1 CAUCAAAGAUG AD- csusucuuUfgCfCfAfucaaagauguL96 935 asCfsaucUfuugauggCfaAfagaagsasu 1294 AUCUUCUUUGCC 1653 1136174.2 AUCAAAGAUGC AD- ususcuuuGfcCfAfUfcaaagaugcuL96 2137 asGfscadTc(Tgn)uugaugGfcAfaagaa 2261 UCUUCUUUGCCA 2385 1297678.1 sgsa UCAAAGAUGCC AD- uscsuuugCfcAfUfCfaaagaugccuL96 2138 asGfsgcdAu(C2p)uuugauGfgCfaaaga 2262 CUUCUUUGCCAU 2386 1297679.1 sasg CAAAGAUGCCA AD- csusuugcCfaUfCfAfaagaugccauL96 2139 asUfsggdCa(Tgn)cuuugaUfgGfcaaag 2263 UUCUUUGCCAUC 2387 1297680.1 sasa UAAAGAUGCCA AD- ususugccAfuCfAfAfagaugccauuL96 2140 asAfsugdGc(Agn)ucuuugAfuGfgcaaa 2264 UCUUUGCCAUCA 2388 1297681.1 sgsa AAGAUGCCAUC AD- ususgccaUfcAfAfAfgaugccaucuL96 2141 asGfsaudGg(C2p)aucuuuGfaUfggcaa 2265 CUUUGCCAUCAA 2389 1297682.1 sasg AGAUGCCAUCC AD- usgsccauCfaAfAfGfaugccauccuL96 2142 asGfsgadTg(G2p)caucuuUfgAfuggca 2266 UUUGCCAUCAAA 2390 1297683.1 sasa GAUGCCAUCCG AD- gscscaucAfaAfGfAfugccauccguL96 2143 asCfsggaUfggcaucuUfuGfauggcsasa 2267 UUGCCAUCAAAG 2391 1297684.1 UAUGCCAUCCG AD- asasaccaAfuGfAfAfcagcaaagcuL96 2144 asGfscudTu(G2p)cuguucAfuUfgguuu 2268 UCAAACCAAUGA 2392 1297685.1 sgsa ACAGCAAAGCA AD- asasccaaUfgAfAfCfagcaaagcauL96 2145 asUfsgcdTu(Tgn)gcuguuCfaUfugguu 2269 CAAACCAAUGAA 2393 1297686.1 susg CAGCAAAGCAU AD- ascscaauGfaAfCfAfgcaaagcauuL96 2146 asAfsugdCu(Tgn)ugcuguUfcAfuuggu 2270 AAACCAAUGAAC 2394 1297687.1 susu AGCAAAGCAUA AD- cscsaaugAfaCfAfGfcaaagcauauL96 2147 asUfsaudGc(Tgn)uugcugUfuCfauugg 2271 AACCAAUGAACA 2395 1297688.1 susu GCAAAGCAUAA AD- csasaugaAfcAfGfCfaaagcauaauL96 2148 asUfsuadTg(C2p)uuugcuGfuUfcauug 2272 ACCAAUGAACAG 2396 1297689.1 sgsu CAAAGCAUAAC AD- asasugaaCfaGfCfAfaagcauaacuL96 2149 asGfsuudAu(G2p)cuuugcUfgUfucauu 2273 CCAAUGAACAGC 2397 1297690.1 sgsg AAAGCAUAACC AD- asusgaacAfgCfAfAfagcauaaccuL96 2150 asGfsgudTa(Tgn)gcuuugCfuGfuucau 2274 CAAUGAACAGCA 2398 1297691.1 susg AAGCAUAACCU AD- usgsaacaGfcAfAfAfgcauaaccuuL96 2151 asAfsgguUfaugcuuuGfcUfguucasusu 2275 AAUGAACAGCAA 2399 1297692.1 AGCAUAACCUU AD- gsasacagCfaAfAfGfcauaaccuuuL96 982 asAfsaggUfuaugcuuUfgCfuguucsasu 1341 AUGAACAGCAAA 1700 1136221.2 GCAUAACCUUG AD- asascagcAfaAfGfCfauaaccuuguL96 983 asCfsaadGg(Tgn)uaugcuUfuGfcuguu 1342 UGAACAGCAAAG 1701 1136222.2 scsa CAUAACCUUGA AD- ascsagcaAfaGfCfAfuaaccuugauL96 2152 asUfscadAg(G2p)uuaugcUfuUfgcugu 2276 GAACAGCAAAGC 2400 1297693.1 susc AUAACCUUGAA AD- csasgcaaAfgCfAfUfaaccuugaauL96 2153 asUfsucdAa(G2p)guuaugCfuUfugcug 2277 AACAGCAAAGCA 2401 1297694.1 susu UAACCUUGAAU AD- asgscaaaGfcAfUfAfaccuugaauuL96 984 asAfsuucAfagguuauGfcUfuugcusgsu 1343 ACAGCAAAGCAU 1702 1136223.2 AACCUUGAAUC AD- gscsaaagCfaUfAfAfccuugaaucuL96 985 asGfsaudTc(Agn)agguuaUfgCfuuugc 1344 CAGCAAAGCAUA 1703 1136224.2 susg ACCUUGAAUCU AD- csasaagcAfuAfAfCfcuugaaucuuL96 986 asAfsgadTu(C2p)aagguuAfuGfcuuug 1345 AGCAAAGCAUAA 1704 1136225.2 scsu CCUUGAAUCUA AD- asasagcaUfaAfCfCfuugaaucuauL96 2154 asUfsagdAu(Tgn)caagguUfaUfgcuuu 2278 GCAAAGCAUAAC 2402 1297695.1 sgsc CUUGAAUCUAU AD- asasgcauAfaCfCfUfugaaucuauuL96 2155 asAfsuagAfuucaaggUfuAfugcuususg 2279 CAAAGCAUAACC 2403 1297696.1 UUGAAUCUAUA AD- asgscauaAfcCfUfUfgaaucuauauL96 2156 asUfsaudAg(Agn)uucaagGfuUfaugcu 2280 AAAGCAUAACCU 2404 1297697.1 susu UGAAUCUAUAC AD- gscsauaaCfcUfUfGfaaucuauacuL96 987 asGfsuauAfgauucaaGfgUfuaugcsusu 1346 AAGCAUAACCUU 1705 1136226.2 GAAUCUAUACU AD- csasuaacCfuUfGfAfaucuauacuuL96 988 asAfsguaUfagauucaAfgGfuuaugscsu 1347 AGCAUAACCUUG 1706 1136227.2 AAUCUAUACUC AD- asusaaccUfuGfAfAfucuauacucuL96 989 asGfsaguAfuagauucAfaGfguuausgsc 1348 GCAUAACCUUGA 1707 1136228.2 AUCUAUACUCA AD- usasaccuUfgAfAfUfcuauacucauL96 990 asUfsgadGu(Agn)uagauuCfaAfgguua 1349 CAUAACCUUGAA 1708 1136229.2 susg UCUAUACUCAA AD- asasccuuGfaAfUfCfuauacucaauL96 991 asUfsugdAg(Tgn)auagauUfcAfagguu 1350 AUAACCUUGAAU 1709 1136230.2 sasu CUAUACUCAAA AD- ascscuugAfaUfCfUfauacucaaauL96 992 asUfsuudGa(G2p)uauagaUfuCfaaggu 1351 UAACCUUGAAUC 1710 1136231.2 susa UAUACUCAAAU AD- cscsuugaAfuCfUfAfuacucaaauuL96 993 asAfsuudTg(Agn)guauagAfuUfcaagg 1352 AACCUUGAAUCU 1711 1136232.2 susu AUACUCAAAUU AD- csusugaaUfcUfAfUfacucaaauuuL96 2157 asAfsaudTu(G2p)aguauaGfaUfucaag 2281 ACCUUGAAUCUA 2405 1297698.1 sgsu UACUCAAAUUU AD- ususgaauCfuAfUfAfcucaaauuuuL96 2158 asAfsaauUfugaguauAfgAfuucaasgsg 2282 CCUUGAAUCUAU 2406 1297699.1 ACUCAAAUUUU AD- gsasaucuAfuAfCfUfcaaauuuuguL96 2159 asCfsaaaAfuuugaguAfuAfgauucsasa 2283 UUGAAUCUAUAC 2407 1297700.1 UCAAAUUUUGC AD- asasucuaUfaCfUfCfaaauuuugcuL96 994 asGfscaaAfauuugagUfaUfagauuscsa 1353 UGAAUCUAUACU 1712 1136233.2 CAAAUUUUGCA AD- asuscuauAfcUfCfAfaauuuugcauL96 995 asUfsgcaAfaauuugaGfuAfuagaususc 1354 GAAUCUAUACUC 1713 1136234.2 AAAUUUUGCAA AD- uscsuauaCfuCfAfAfauuuugcaauL96 2160 asUfsugcAfaaauuugAfgUfauagasusu 2284 AAUCUAUACUCA 2408 1297701.1 AAUUUUGCAAU AD- csusauacUfcAfAfAfuuuugcaauuL96 2161 asAfsuudGc(Agn)aaauuuGfaGfuauag 2285 AUCUAUACUCAA 2409 1297702.1 sasu AUUUUGCAAUG AD- usasuacuCfaAfAfUfuuugcaauguL96 2162 asCfsaudTg(C2p)aaaauuUfgAfguaua 2286 UCUAUACUCAAA 2410 1297703.1 sgsa UUUUGCAAUGA AD- asusacucAfaAfUfUfuugcaaugauL96 2163 asUfscadTu(G2p)caaaauUfuGfaguau 2287 CUAUACUCAAAU 2411 1297704.1 sasg UUUGCAAUGAG AD- usascucaAfaUfUfUfugcaaugaguL96 2164 asCfsucaUfugcaaaaUfuUfgaguasusa 2288 UAUACUCAAAUU 2412 1297705.1 UUGCAAUGAGG AD- ascsucaaAfuUfUfUfgcaaugagguL96 2165 asCfscucAfuugcaaaAfuUfugagusasu 2289 AUACUCAAAUUU 2413 1297706.1 UGCAAUGAGGC AD- csuscaaaUfuUfUfGfcaaugaggcuL96 2166 asGfsccdTc(Agn)uugcaaAfaUfuugag 2290 UACUCAAAUUUU 2414 1297707.1 susa GCAAUGAGGCA AD- uscsaaauUfuUfGfCfaaugaggcauL96 2167 asUfsgcdCu(C2p)auugcaAfaAfuuuga 2291 ACUCAAAUUUUG 2415 1297708.1 sgsu CAAUGAGGCAG AD- csasaauuUfuGfCfAfaugaggcaguL96 2168 asCfsugdCc(Tgn)cauugcAfaAfauuug 2292 CUCAAAUUUUGC 2416 1297709.1 sasg AAUGAGGCAGU AD- asasauuuUfgCfAfAfugaggcaguuL96 2169 asAfscudGc(C2p)ucauugCfaAfaauuu 2293 UCAAAUUUUGCA 2417 1297710.1 sgsa AUGAGGCAGUG AD- asasuuuuGfcAfAfUfgaggcaguguL96 2170 asCfsacdTg(C2p)cucauuGfcAfaaauu 2294 CAAAUUUUGCAA 2418 1297711.1 susg UGAGGCAGUGG AD- asusuuugCfaAfUfGfaggcagugguL96 2171 asCfscacUfgccucauUfgCfaaaaususu 2295 AAAUUUUGCAAU 2419 1297712.1 GAGGCAGUGGG AD- csuscuccaagUfAfUfgaucgucuuL96 2172 asdAsgadCgdAucaudAcUfuggagagscs 2296 UGCUCUCCAAGU 1476 1395794.1 g GAUGAUCGUCU AD- asgsgaucucUfCfUfcagaguauuuL96 2173 asdAsaudAc(Tgn)cugadGadGadGaucc 2297 CCAGGAUCUCUC 1562 1395797.1 usgsg UCAGAGUAUUA AD- csasgugaugCfUfCfuccaaguauuL96 2174 asdAsuadCudTggagdAgCfaucacugsus 2298 CACAGUGAUGCU 2305 1395803.1 g CUCCAAGUAUG AD- cscsaaguauGfAfUfcgucugcaguL96 2175 asdCsugdCadGacgadTcAfuacuuggsas 2299 CUCCAAGUAUGA 2312 1395805.1 g UCGUCUGCAGA AD- gsusuuguuuCfAfUfuuccgcugauL96 2176 asdTscadGc(G2p)gaaadTgAfaacaaac 2300 UUGUUUGUUUCA 2339 1395807.1 sgsg UUUUCCGCUGA AD- asascacccaGfGfAfucucucucauL96 2177 asdTsgadGa(G2p)agaudCcUfggguguu 2301 GGAACACCCAGG 2364 1395811.1 scsc AUCUCUCUCAG AD- gsgsguuuguUfudGUfuucauuucuL96 2178 asdGsaadAudGaaacdAaAfcaaacccsus 2302 CAGGGUUUGUUU 1532 1395816.1 g GUUUCAUUUCC AD- gsasguauuuCfUfCfagcauucaauL96 2179 asdTsugdAadTgcugdAgAfaauacucscs 2303 GGGAGUAUUUCU 1519 1395823.1 c CAGCAUUCAAG

TABLE 8 Xanthine Dehydrogenase dsRNA Agent In Vitro Single Dose Screens in Primary Human Hepatocytes PHH 500 nM 100 nM 10 nM 0.1 nM % of Avg % of Avg % of Avg % of Avg Message ST Message ST Message ST Message ST DuplexID Remaining DEV Remaining DEV Remaining DEV Remaining DEV AD- 69.5 32.5 49.2 13.7 87.8 12.8 68.7 9.1 1135990.2 AD- 36.5 5.0 47.2 4.8 73.1 16.7 85.6 18.1 1135991.2 AD- 34.7 7.4 36.6 15.2 80.0 23.2 76.5 5.6 1297597.1 AD- 29.7 8.7 37.2 5.8 65.1 13.4 51.9 27.4 1297598.1 AD- 47.9 15.5 59.7 6.3 110.0 22.3 74.3 23.6 1297599.1 AD- 37.7 11.6 60.6 5.6 87.2 4.5 67.7 15.9 1297600.1 AD- 43.3 7.9 61.2 19.4 91.8 11.2 93.9 12.0 1297601.1 AD- 34.0 6.5 31.7 16.9 64.5 31.3 90.8 26.9 1135992.2 AD- 54.4 5.9 60.8 13.9 107.4 31.0 111.3 5.6 1135993.2 AD- 84.8 8.6 95.6 8.2 109.1 15.8 119.7 11.0 1135994.2 AD- 59.0 4.3 75.2 14.3 94.9 26.1 95.8 15.0 1135995.2 AD- 31.1 4.3 50.8 7.1 63.5 16.7 80.2 21.7 1135996.2 AD- 74.7 7.3 73.2 12.7 104.5 7.3 84.3 21.0 1297602.1 AD- 91.4 9.7 142.7 74.0 117.3 17.7 110.9 14.0 1297603.1 AD- 50.1 10.9 57.4 8.8 95.0 29.8 120.8 28.8 1297604.1 AD- 30.8 4.7 35.7 25.5 86.0 26.2 140.8 33.5 1297605.1 AD- 86.7 18.6 68.4 12.6 85.1 7.2 89.8 6.6 1297606.1 AD- 47.3 3.0 56.6 18.7 70.1 7.6 103.3 8.5 1297607.1 AD- 47.2 7.7 49.6 10.5 65.7 9.5 84.7 20.3 1297608.1 AD- 63.2 6.4 61.0 7.5 96.6 16.7 112.8 22.7 1297609.1 AD- 60.3 9.7 78.9 10.5 104.7 12.3 118.0 17.4 1297610.1 AD- 65.7 14.9 70.0 9.6 88.0 10.6 116.1 13.8 1297611.1 AD- 35.8 3.7 27.7 7.9 78.2 13.9 138.3 0.0 1136037.2 AD- 37.6 1.1 26.3 10.8 77.8 8.7 86.1 27.4 1136038.2 AD- 42.2 7.7 75.4 8.1 88.3 9.9 96.2 22.8 1136039.2 AD- 63.0 9.8 89.8 7.8 86.4 21.0 89.9 11.6 1136040.2 AD- 83.0 16.4 70.0 21.5 105.9 21.4 98.7 5.0 1136041.2 AD- 84.2 17.1 83.6 5.9 106.2 14.6 103.2 7.8 1297612.1 AD- 52.7 20.3 89.6 28.1 97.3 15.1 85.6 12.9 1297613.1 AD- 72.6 14.9 70.6 30.7 74.5 17.3 96.5 12.5 1297614.1 AD- 73.9 6.0 94.3 31.6 96.2 10.0 113.8 37.2 1297615.1 AD- 69.5 12.7 89.8 4.4 103.9 23.4 102.4 31.5 1297616.1 AD- 62.5 17.7 53.4 8.0 79.5 9.8 87.2 5.8 1297617.1 AD- 48.2 0.0 76.0 12.7 106.7 35.5 81.6 10.8 1297618.1 AD- 90.3 21.9 81.4 9.4 106.3 14.9 96.6 24.9 1297619.1 AD- 48.1 11.5 47.7 4.6 90.3 28.9 111.2 26.6 1297620.1 AD- 43.9 12.6 54.0 12.5 88.6 18.3 84.4 20.6 1297621.1 AD- 52.2 17.4 65.1 23.1 103.5 8.3 105.1 25.5 1297622.1 AD- 79.5 3.9 78.2 21.8 107.4 6.1 107.4 14.9 1297623.1 AD- 46.9 22.9 66.8 22.5 94.6 10.2 102.2 24.4 1297624.1 AD- 71.6 25.9 88.4 16.0 84.4 34.9 110.2 31.1 1297625.1 AD- 74.1 21.9 61.7 12.1 89.4 14.6 98.4 13.2 1297626.1 AD- 47.9 27.7 62.8 27.3 89.2 16.2 102.8 34.7 1136050.2 AD- 42.7 21.0 43.9 10.8 90.9 28.5 112.4 29.7 1297627.1 AD- 27.2 2.8 40.3 11.7 63.8 22.0 73.5 28.0 1297628.1 AD- 37.4 5.3 44.9 12.0 45.0 11.8 91.8 11.6 1136051.2 AD- 41.2 10.0 44.4 11.2 76.3 4.5 70.5 28.6 1136052.2 AD- 38.7 7.0 68.6 8.4 82.4 38.3 129.7 35.5 1136053.2 AD- 53.8 17.9 57.9 11.7 89.1 15.3 137.6 34.8 1297629.1 AD- 58.7 23.3 85.6 17.4 96.6 21.8 70.5 33.0 1297630.1 AD- 53.3 22.5 62.8 10.3 85.7 25.7 107.7 24.2 1136054.2 AD- 48.2 15.8 80.4 15.6 107.1 16.3 110.5 25.1 1297631.1 AD- 42.1 7.2 43.0 10.0 96.2 9.7 103.3 1.0 1297632.1 AD- 43.2 8.9 90.4 11.0 96.5 20.0 93.0 6.0 1297633.1 AD- 54.6 15.4 88.7 21.6 85.9 16.7 106.6 12.6 1297634.1 AD- 67.7 14.5 74.6 16.9 102.2 24.8 84.7 26.6 1297635.1 AD- 80.8 15.9 93.2 14.2 106.6 23.4 120.7 34.7 1297636.1 AD- 40.7 4.8 73.3 18.4 72.2 11.8 105.2 17.1 1297637.1 AD- 79.9 14.7 98.6 15.9 120.9 20.9 97.7 31.5 1297638.1 AD- 41.6 9.7 84.5 15.6 107.2 11.6 128.2 9.5 1297639.1 AD- 82.8 18.5 82.7 23.1 97.3 15.4 199.9 12.3 1297640.1 AD- 74.2 6.0 90.5 31.3 92.7 6.3 101.5 20.7 1297641.1 AD- 50.8 13.4 87.0 13.3 82.3 6.7 101.5 15.8 1297642.1 AD- 44.1 6.1 79.7 n/a 102.6 15.2 120.1 24.2 1297643.1 AD- 73.8 19.0 87.5 23.9 80.4 22.3 99.8 36.1 1297644.1 AD- 80.8 26.6 100.5 15.6 92.8 31.6 103.2 15.6 1297645.1 AD- 54.6 20.8 125.2 25.4 135.6 21.5 114.8 14.6 1136073.2 AD- 72.1 n/a 79.6 7.4 88.6 19.7 129.9 3.6 1136074.2 AD- 38.2 9.0 52.3 15.5 90.8 28.0 76.2 15.0 1136075.2 AD- 82.6 15.6 114.4 55.2 116.3 29.6 116.6 26.6 1136076.2 AD- 58.6 19.5 72.0 8.4 82.5 19.2 102.0 33.4 1136077.2 AD- 86.7 5.2 129.5 18.3 118.8 14.8 108.6 19.1 1136078.2 AD- 69.8 17.8 123.8 58.6 93.4 10.9 137.9 28.8 1297646.1 AD- 58.3 9.4 56.0 16.9 122.6 18.1 119.8 18.6 1297647.1 AD- 86.3 12.5 90.3 0.0 95.0 23.8 140.1 35.6 1297648.1 AD- 42.0 13.5 70.2 14.8 92.5 15.1 90.3 4.5 1297649.1 AD- 68.8 6.7 80.5 18.9 103.1 8.2 121.7 8.8 1297650.1 AD- 68.3 5.8 99.0 28.3 73.4 14.4 105.5 22.0 1297651.1 AD- 99.0 30.9 109.3 18.4 111.7 25.7 103.6 8.6 1297652.1 AD- 53.3 15.2 88.9 62.0 111.2 27.5 95.2 16.1 1297653.1 AD- 126.3 16.6 110.5 19.0 105.7 10.3 91.4 8.1 1297654.1 AD- 116.3 11.7 124.4 22.3 87.0 8.5 126.4 11.7 1297655.1 AD- 83.3 14.8 65.2 6.7 101.1 18.3 90.7 18.0 1297656.1 AD- 22.0 8.2 51.6 49.2 62.9 8.9 107.5 30.9 1297657.1 AD- 71.1 3.5 78.2 14.6 103.9 10.2 106.4 11.4 1297658.1 AD- 63.9 18.4 73.0 22.4 89.1 10.1 95.7 18.8 1297659.1 AD- 54.6 13.9 70.9 44.2 101.9 19.1 90.4 19.2 1297660.1 AD- 67.9 35.5 61.7 14.7 91.0 23.6 81.0 33.3 1297661.1 AD- 74.3 15.3 69.9 16.7 97.1 21.9 79.0 19.9 1297662.1 AD- 77.0 24.1 36.6 7.8 85.9 25.6 103.4 7.0 1297663.1 AD- 57.0 0.6 65.1 13.3 47.7 8.6 n/d n/d 1136082.2 AD- 82.2 8.9 76.8 26.0 41.1 19.0 n/d n/d 1136083.2 AD- 81.8 7.2 87.4 13.5 50.2 8.6 n/d n/d 1136084.2 AD- 98.6 21.4 102.9 38.2 75.8 30.0 n/d n/d 1136085.2 AD- 91.7 33.5 102.1 10.8 67.5 8.4 n/d n/d 1136086.2 AD- 87.2 23.0 63.5 1.9 54.8 12.9 n/d n/d 1136087.2 AD- 99.5 11.4 73.3 24.0 69.1 7.2 n/d n/d 1136088.2 AD- 87.8 11.4 96.4 43.3 57.6 18.6 n/d n/d 1136089.2 AD- 101.3 15.8 116.1 14.8 74.6 20.4 n/d n/d 1136090.2 AD- 73.8 12.3 84.0 6.5 65.7 3.8 n/d n/d 1297664.1 AD- 52.3 14.5 58.2 11.3 55.3 15.3 n/d n/d 1136091.2 AD- 62.4 6.9 71.3 10.6 55.6 26.6 n/d n/d 1136092.2 AD- 62.9 10.6 75.5 6.9 62.1 11.5 n/d n/d 1297665.1 AD- 54.8 13.8 59.6 15.2 51.1 7.3 n/d n/d 1297666.1 AD- 65.8 5.0 66.6 24.9 63.3 14.4 n/d n/d 1297667.1 AD- 66.2 10.2 68.8 3.7 54.6 15.8 n/d n/d 1297668.1 AD- 73.4 9.4 72.2 2.5 65.9 13.6 n/d n/d 1297669.1 AD- 57.1 7.3 73.4 19.6 61.2 8.5 n/d n/d 1297670.1 AD- 63.1 13.1 94.9 24.4 88.0 6.3 n/d n/d 1297671.1 AD- 123.7 12.5 124.9 10.9 101.4 27.4 n/d n/d 1297672.1 AD- 60.0 10.8 66.5 4.4 71.3 16.0 n/d n/d 1297673.1 AD- 96.2 26.1 111.6 8.5 110.5 9.5 n/d n/d 1297674.1 AD- 75.1 3.9 95.9 3.3 73.9 48.8 n/d n/d 1297675.1 AD- 56.0 6.6 106.5 19.2 55.0 9.8 n/d n/d 1136159.2 AD- 69.8 15.3 84.4 6.2 81.7 8.5 n/d n/d 1136160.2 AD- 75.8 24.6 96.5 20.6 93.1 5.0 n/d n/d 1136161.2 AD- 73.6 8.4 74.9 19.7 76.2 10.7 n/d n/d 1136162.2 AD- 62.4 14.7 86.2 18.7 55.2 20.3 n/d n/d 1136163.2 AD- 59.9 11.9 65.8 22.6 88.1 36.6 n/d n/d 1297676.1 AD- 94.5 11.4 100.5 14.2 92.6 23.3 n/d n/d 1136164.2 AD- 69.2 16.2 71.0 6.2 64.7 9.2 n/d n/d 1136165.2 AD- 47.1 9.8 56.5 21.4 70.8 5.0 n/d n/d 1136166.2 AD- 107.0 14.0 102.6 27.8 86.2 9.9 n/d n/d 1136167.2 AD- 104.9 22.3 86.7 26.5 98.1 15.2 n/d n/d 1136168.2 AD- 64.4 21.6 54.5 16.6 67.6 15.3 n/d n/d 1136169.2 AD- 64.6 14.6 66.7 12.0 80.8 12.9 n/d n/d 1136170.2 AD- 100.8 17.6 111.3 13.2 106.0 21.1 n/d n/d 1136171.2 AD- 72.0 14.2 88.1 23.7 69.4 28.7 n/d n/d 1136172.2 AD- 97.4 26.7 103.0 57.7 78.0 1.1 n/d n/d 1136173.2 AD- 96.1 26.8 106.2 38.5 93.4 26.2 n/d n/d 1297677.1 AD- 75.2 20.0 96.4 10.3 100.4 10.2 n/d n/d 1136174.2 AD- 118.3 19.4 111.5 20.6 103.2 15.4 n/d n/d 1297678.1 AD- 116.0 12.2 112.6 31.9 111.8 7.4 n/d n/d 1297679.1 AD- 90.0 27.9 95.8 29.6 105.8 20.4 n/d n/d 1297680.1 AD- 105.9 13.9 109.2 10.6 111.6 16.4 n/d n/d 1297681.1 AD- 77.2 14.6 92.4 4.5 76.6 16.7 n/d n/d 1297682.1 AD- 64.0 14.8 90.5 16.1 71.8 14.7 n/d n/d 1297683.1 AD- 87.4 2.0 87.7 15.4 71.7 31.3 n/d n/d 1297684.1 AD- 90.2 15.1 94.3 5.2 113.9 38.3 n/d n/d 1297685.1 AD- 94.7 9.8 80.8 7.5 117.1 21.2 n/d n/d 1297686.1 AD- 58.8 11.8 79.6 2.4 106.6 18.3 n/d n/d 1297687.1 AD- 82.2 5.0 78.0 26.0 78.6 6.3 n/d n/d 1297688.1 AD- 59.7 21.1 86.7 1.7 74.8 25.1 n/d n/d 1297689.1 AD- 74.9 26.2 86.5 11.0 74.1 25.1 n/d n/d 1297690.1 AD- 88.5 22.1 103.7 14.2 90.9 18.6 n/d n/d 1297691.1 AD- 105.8 21.4 103.4 26.9 86.1 8.3 n/d n/d 1297692.1 AD- 67.9 32.7 72.6 12.3 100.3 10.7 n/d n/d 1136221.2 AD- 94.3 12.7 127.7 28.6 93.0 4.6 n/d n/d 1136222.2 AD- 77.2 25.7 97.6 22.6 113.9 12.6 n/d n/d 1297693.1 AD- 81.4 10.1 109.5 5.5 94.1 9.6 n/d n/d 1297694.1 AD- 64.0 8.4 61.8 21.1 98.3 20.4 n/d n/d 1136223.2 AD- 63.4 2.3 62.9 15.0 67.7 25.1 n/d n/d 1136224.2 AD- 87.1 19.2 92.7 11.0 63.9 14.1 n/d n/d 1136225.2 AD- 80.2 16.5 77.9 3.4 100.9 21.4 n/d n/d 1297695.1 AD- 71.7 12.4 70.7 21.0 90.1 16.0 n/d n/d 1297696.1 AD- 108.1 5.3 89.0 12.7 119.6 13.2 n/d n/d 1297697.1 AD- 92.0 4.1 95.4 29.2 100.4 14.5 n/d n/d 1136226.2 AD- 60.5 15.7 91.0 1.8 74.5 17.4 n/d n/d 1136227.2 AD- 61.2 4.0 77.8 18.6 57.0 13.0 n/d n/d 1136228.2 AD- 78.3 12.0 80.1 8.3 89.1 18.7 n/d n/d 1136229.2 AD- 92.2 19.3 106.9 14.0 82.9 14.7 n/d n/d 1136230.2 AD- 96.9 12.5 85.5 14.0 95.5 14.0 n/d n/d 1136231.2 AD- 79.5 8.3 73.1 24.7 87.6 15.3 n/d n/d 1136232.2 AD- 76.3 7.0 79.1 23.3 101.6 21.5 n/d n/d 1297698.1 AD- 44.5 12.0 67.5 17.5 87.0 19.1 n/d n/d 1297699.1 AD- 86.3 19.6 82.6 19.5 58.2 8.5 n/d n/d 1297700.1 AD- 86.6 14.4 80.5 9.5 73.6 11.6 n/d n/d 1136233.2 AD- 85.4 15.2 77.1 13.1 80.3 17.2 n/d n/d 1136234.2 AD- 79.3 8.4 84.4 14.6 72.0 24.3 n/d n/d 1297701.1 AD- 100.3 3.2 83.1 11.3 95.1 14.0 n/d n/d 1297702.1 AD- 78.0 26.3 117.3 16.3 112.4 34.5 n/d n/d 1297703.1 AD- 85.1 8.4 95.1 24.5 111.8 37.5 n/d n/d 1297704.1 AD- 74.4 17.9 84.1 56.6 103.6 6.6 n/d n/d 1297705.1 AD- 101.6 28.1 68.8 32.4 47.8 6.1 n/d n/d 1297706.1 AD- 94.1 21.1 86.9 8.5 62.1 23.6 n/d n/d 1297707.1 AD- 80.7 18.7 76.0 12.5 82.0 13.4 n/d n/d 1297708.1 AD- 78.1 8.8 74.3 26.0 72.2 18.5 n/d n/d 1297709.1 AD- 68.9 12.3 79.7 20.2 79.0 14.7 n/d n/d 1297710.1 AD- 78.2 17.6 95.3 27.3 72.0 9.0 n/d n/d 1297711.1 AD- 86.2 10.5 88.8 16.4 90.9 26.1 n/d n/d 1297712.1

TABLE 9 Xanthine Dehydrogenase dsRNA Agent In Vitro Single Dose Screens in Primary Human Hepatocytes PHH 500 nM 100 nM 10 nM 0.1 nM % of Avg % of Avg % of Avg % of Avg Message ST Message ST Message ST Message ST DuplexID Remaining DEV Remaining DEV Remaining DEV Remaining DEV AD- 77.7 14.6 46.5 24.5 40.3 26.2 53.9 33.2 1135990.2 AD- 60.7 15.3 35.9 16.3 41.1 14.3 75.1 36.6 1135991.2 AD- 56.8 25.9 36.9 11.8 72.0 45.0 51.9 24.4 1297597.1 AD- 44.7 28.8 69.4 28.7 48.5 26.4 79.8 25.9 1297598.1 AD- 83.4 29.1 97.6 37.6 50.7 15.3 78.5 20.8 1297599.1 AD- 88.1 21.6 43.5 12.7 45.6 7.3 70.4 21.5 1297600.1 AD- 57.5 40.4 46.3 11.3 49.5 9.4 72.6 21.3 1297601.1 AD- 50.9 17.5 43.7 6.5 33.5 7.4 38.5 25.0 1135992.2 AD- 58.0 16.6 79.5 21.3 63.4 48.2 71.4 39.9 1135993.2 AD- 117.8 14.1 81.7 22.1 103.8 30.2 102.9 39.5 1135994.2 AD- 131.8 27.0 78.5 22.8 152.4 18.4 120.4 28.1 1135995.2 AD- 47.0 16.3 49.9 19.2 115.5 18.3 89.3 22.1 1135996.2 AD- 48.7 31.0 106.6 39.7 97.9 27.4 82.2 16.9 1297602.1 AD- 108.1 42.1 88.7 18.4 81.5 35.9 72.8 14.8 1297603.1 AD- 62.9 36.3 66.1 16.0 69.7 15.9 90.1 9.7 1297604.1 AD- 49.1 14.5 45.4 14.3 69.5 44.7 48.9 16.4 1297605.1 AD- 74.2 20.6 79.3 13.2 43.3 7.8 63.1 7.3 1297606.1 AD- 68.6 35.0 63.8 30.0 44.3 10.3 99.1 19.8 1297607.1 AD- 75.7 39.0 85.4 38.0 85.7 21.5 87.4 11.7 1297608.1 AD- 114.4 26.2 99.9 35.8 98.8 16.2 95.8 34.8 1297609.1 AD- 87.7 8.0 73.1 31.8 72.3 22.7 102.1 23.6 1297610.1 AD- 93.5 18.0 71.8 20.5 57.3 26.1 81.0 15.2 1297611.1 AD- 53.2 8.0 48.1 22.9 65.4 8.6 52.6 22.2 1136037.2 AD- 58.1 25.6 26.4 3.6 37.2 13.9 67.7 21.8 1136038.2 AD- 72.8 19.0 54.2 30.3 75.6 28.9 82.3 30.9 1136039.2 AD- 122.9 44.6 123.6 56.7 89.2 31.0 67.6 8.7 1136040.2 AD- 123.5 38.0 138.3 31.7 85.7 28.1 131.5 22.0 1136041.2 AD- 132.7 41.5 137.2 41.0 107.2 34.9 123.0 17.7 1297612.1 AD- 73.8 31.3 88.3 31.9 83.8 24.7 98.0 22.7 1297613.1 AD- 79.8 12.9 103.4 36.9 67.2 N/A 68.9 30.2 1297614.1 AD- 123.9 44.3 94.2 69.5 86.7 40.1 96.3 43.2 1297615.1 AD- 106.0 35.0 85.7 41.7 84.4 29.8 112.5 12.9 1297616.1 AD- 60.7 22.5 99.8 66.2 85.9 46.1 141.1 32.9 1297617.1 AD- 111.7 43.5 135.2 44.2 98.6 46.9 109.9 21.0 1297618.1 AD- 110.0 14.8 133.3 29.2 126.3 42.2 120.9 39.1 1297619.1 AD- 84.4 26.6 135.3 39.8 91.7 15.7 114.7 15.3 1297620.1 AD- 60.2 20.8 77.7 7.4 88.2 32.9 85.4 15.5 1297621.1 AD- 77.1 19.3 96.8 29.0 105.2 55.1 69.2 37.5 1297622.1 AD- 85.5 40.2 82.6 28.3 148.1 48.2 92.7 42.3 1297623.1 AD- 44.0 5.7 61.2 9.0 91.6 1.6 104.6 69.2 1297624.1 AD- 117.2 42.9 121.1 41.8 131.3 43.9 120.6 47.1 1297625.1 AD- 112.4 26.9 118.6 66.3 134.0 24.6 145.2 41.0 1297626.1 AD- 62.9 27.3 74.8 13.0 149.8 50.5 142.6 26.7 1136050.2 AD- 95.4 39.7 79.4 26.9 110.3 32.9 129.4 16.4 1297627.1 AD- 79.7 27.1 59.1 15.5 68.3 19.3 96.1 16.2 1297628.1 AD- 39.1 21.5 34.8 18.9 53.6 49.8 62.8 16.7 1136051.2 AD- 77.1 17.9 65.1 44.0 95.6 49.0 98.3 36.1 1136052.2 AD- 76.2 40.2 100.9 6.9 108.5 44.0 101.0 11.9 1136053.2 AD- 90.8 46.5 97.0 23.5 108.3 33.9 122.0 41.3 1297629.1 AD- 119.0 29.3 141.6 67.2 151.1 46.4 138.4 32.1 1297630.1 AD- 123.4 8.4 104.4 32.9 117.1 27.4 113.9 15.5 1136054.2 AD- 78.3 9.5 118.5 49.3 127.7 20.5 116.4 36.1 1297631.1 AD- 53.1 10.2 35.8 9.4 93.9 17.2 59.1 19.8 1297632.1 AD- 80.5 31.1 68.3 50.4 102.5 61.9 91.1 8.7 1297633.1 AD- 107.7 25.2 101.7 40.4 112.3 18.7 137.5 36.5 1297634.1 AD- 126.7 22.6 138.4 77.3 142.0 40.9 132.4 15.3 1297635.1 AD- 126.6 23.1 169.9 18.8 157.9 51.9 143.2 13.6 1297636.1 AD- 92.1 39.5 166.8 55.2 131.7 39.1 134.5 44.5 1297637.1 AD- 148.5 15.3 131.7 64.4 142.3 44.7 127.2 11.1 1297638.1 AD- 106.8 43.2 115.6 12.7 152.9 32.2 112.3 14.5 1297639.1 AD- 95.4 33.6 74.2 35.4 80.9 39.9 65.6 24.0 1297640.1 AD- 62.6 40.2 75.2 38.8 101.0 40.2 91.7 35.7 1297641.1 AD- 94.0 27.1 87.7 25.2 107.6 42.4 100.7 26.6 1297642.1 AD- 171.8 19.3 163.5 59.8 148.8 39.6 144.6 47.4 1297643.1 AD- 120.4 27.8 116.4 74.3 159.7 41.0 146.9 35.2 1297644.1 AD- 132.7 16.5 124.9 68.2 135.4 47.3 131.8 14.5 1297645.1 AD- 107.0 29.3 84.4 19.5 110.2 40.6 87.2 13.5 1136073.2 AD- 64.0 37.4 75.5 9.9 77.0 43.9 46.7 17.6 1136074.2 AD- 57.4 28.4 55.8 14.3 75.8 54.4 105.1 2.4 1136075.2 AD- 134.2 49.0 77.6 41.8 113.1 45.4 129.4 22.5 1136076.2 AD- 113.0 39.9 120.7 46.9 137.8 37.8 142.3 14.0 1136077.2 AD- 131.8 55.1 178.5 56.6 162.1 67.7 123.9 5.3 1136078.2 AD- 93.7 34.8 136.6 47.1 166.9 10.2 131.9 14.5 1297646.1 AD- 93.6 35.1 64.8 30.2 114.4 17.8 102.2 31.1 1297647.1 AD- 93.2 37.3 71.0 18.1 105.6 16.3 50.9 6.0 1297648.1 AD- 42.1 9.3 48.7 24.7 42.6 20.2 53.8 11.5 1297649.1 AD- 99.4 15.2 152.2 18.6 135.9 21.5 121.5 16.7 1297650.1 AD- 95.1 26.3 108.1 38.1 85.9 24.5 102.8 21.6 1297651.1 AD- 154.3 23.3 135.2 52.7 139.4 29.3 132.3 10.5 1297652.1 AD- 83.7 34.5 76.3 32.5 138.2 47.7 140.0 10.9 1297653.1 AD- 117.1 41.4 71.0 25.4 141.1 68.1 90.2 6.9 1297654.1 AD- 86.2 32.0 94.8 23.8 108.4 27.9 83.0 19.5 1297655.1 AD- 82.2 45.6 59.4 28.2 75.7 44.5 38.7 16.3 1297656.1 AD- 26.2 3.2 23.2 11.8 54.0 51.9 55.2 27.3 1297657.1 AD- 72.2 16.6 82.9 48.2 72.9 51.9 84.7 25.9 1297658.1 AD- 64.9 27.0 60.3 13.8 91.4 40.8 66.0 24.7 1297659.1 AD- 63.0 27.9 98.7 51.5 77.7 69.3 93.3 33.9 1297660.1 AD- 39.7 24.5 68.4 30.8 69.3 32.7 93.8 25.5 1297661.1 AD- 58.6 25.8 42.1 12.7 48.2 30.1 64.3 16.4 1297662.1 AD- 50.3 11.1 24.4 8.9 39.7 19.9 53.4 15.9 1297663.1 AD- 16.1 10.8 64.9 27.2 81.5 20.2 81.5 20.2 1136082.2 AD- 51.2 13.6 72.2 16.5 102.0 14.1 102.0 14.1 1136083.2 AD- 55.5 32.4 98.3 30.3 112.8 22.6 112.8 22.6 1136084.2 AD- 57.8 35.2 84.0 23.8 124.3 4.7 124.3 4.7 1136085.2 AD- 79.6 24.1 112.1 8.6 92.6 29.1 92.6 29.1 1136086.2 AD- 62.4 7.5 80.3 19.8 93.5 13.7 93.5 13.7 1136087.2 AD- 64.3 10.4 96.8 8.3 94.2 30.4 94.2 30.4 1136088.2 AD- 40.8 31.0 55.3 15.4 88.4 23.2 88.4 23.2 1136089.2 AD- 61.4 30.3 131.1 32.5 137.3 33.0 137.3 33.0 1136090.2 AD- 96.5 25.0 100.0 21.1 135.0 35.2 135.0 35.2 1297664.1 AD- 74.3 29.0 84.0 28.1 95.3 11.3 95.3 11.3 1136091.2 AD- 83.0 31.0 66.2 14.4 108.0 7.5 108.0 7.5 1136092.2 AD- 62.2 25.2 68.7 20.6 88.4 9.3 88.4 9.3 1297665.1 AD- 51.2 13.0 47.1 7.5 78.9 12.0 78.9 12.0 1297666.1 AD- 55.4 18.2 67.5 21.1 70.5 10.2 70.5 10.2 1297667.1 AD- 50.9 18.1 70.6 15.2 98.5 19.5 98.5 19.5 1297668.1 AD- 52.7 18.5 99.2 21.6 103.8 19.6 103.8 19.6 1297669.1 AD- 80.5 26.4 88.7 32.0 113.9 8.1 113.9 8.1 1297670.1 AD- 71.4 24.3 71.7 7.8 98.7 7.6 98.7 7.6 1297671.1 AD- 103.0 4.8 97.6 3.4 119.4 33.0 119.4 33.0 1297672.1 AD- 47.8 19.5 60.8 21.7 98.4 10.1 98.4 10.1 1297673.1 AD- 96.5 17.6 86.3 14.8 93.4 11.4 93.4 11.4 1297674.1 AD- 42.9 9.7 72.8 16.2 80.5 10.6 80.5 10.6 1297675.1 AD- 76.7 20.7 65.0 25.8 107.4 22.5 107.4 22.5 1136159.2 AD- 74.6 15.1 77.2 20.1 131.3 1.6 131.3 1.6 1136160.2 AD- 81.1 33.0 80.1 20.6 103.6 26.3 103.6 26.3 1136161.2 AD- 66.4 12.3 88.3 37.3 124.8 13.6 124.8 13.6 1136162.2 AD- 69.6 18.4 50.6 8.3 89.5 11.6 89.5 11.6 1136163.2 AD- 55.7 30.9 51.5 14.1 78.1 17.8 78.1 17.8 1297676.1 AD- 41.7 11.4 79.9 27.9 72.8 23.5 72.8 23.5 1136164.2 AD- 57.4 18.9 91.6 15.6 109.6 20.4 109.6 20.4 1136165.2 AD- 70.7 19.9 62.2 24.4 110.2 28.8 110.2 28.8 1136166.2 AD- 117.2 19.8 100.5 32.0 145.2 13.9 145.2 13.9 1136167.2 AD- 72.9 11.4 69.2 12.1 115.2 25.9 115.2 25.9 1136168.2 AD- 52.4 6.6 39.9 7.6 105.5 15.2 105.5 15.2 1136169.2 AD- 60.2 13.0 58.5 34.9 88.9 22.0 88.9 22.0 1136170.2 AD- 93.8 9.6 75.3 11.1 72.9 15.5 72.9 15.5 1136171.2 AD- 55.6 25.3 65.7 17.7 83.9 21.8 83.9 21.8 1136172.2 AD- 79.0 36.1 120.7 34.2 127.9 14.1 127.9 14.1 1136173.2 AD- 89.9 17.7 100.3 37.6 90.2 34.0 90.2 34.0 1297677.1 AD- 84.7 10.4 94.9 28.6 113.4 16.5 113.4 16.5 1136174.2 AD- 110.9 29.0 76.8 13.1 140.0 13.6 140.0 13.6 1297678.1 AD- 115.4 5.5 120.9 35.8 122.2 23.8 122.2 23.8 1297679.1 AD- 98.6 13.3 102.9 31.1 103.7 0.8 103.7 0.8 1297680.1 AD- 97.1 14.8 84.5 27.3 95.6 17.1 95.6 17.1 1297681.1 AD- 95.1 15.9 111.8 16.7 116.9 32.7 116.9 32.7 1297682.1 AD- 101.0 20.4 85.3 21.6 128.9 12.9 128.9 12.9 1297683.1 AD- 92.9 24.8 69.1 12.3 109.7 21.6 109.7 21.6 1297684.1 AD- 54.7 12.1 67.4 27.5 103.5 5.8 103.5 5.8 1297685.1 AD- 81.4 10.3 98.4 30.3 115.9 14.0 115.9 14.0 1297686.1 AD- 62.0 8.1 63.0 18.0 102.1 8.7 102.1 8.7 1297687.1 AD- 41.4 12.5 49.4 10.4 74.8 20.3 74.8 20.3 1297688.1 AD- 61.0 16.6 51.0 12.1 58.3 8.2 58.3 8.2 1297689.1 AD- 64.4 28.9 89.6 27.5 116.6 25.3 116.6 25.3 1297690.1 AD- 75.7 22.0 99.7 24.9 123.5 26.7 123.5 26.7 1297691.1 AD- 130.0 27.2 100.0 30.0 127.8 23.3 127.8 23.3 1297692.1 AD- 86.4 21.5 72.9 42.0 107.5 5.2 107.5 5.2 1136221.2 AD- 142.1 15.6 109.6 38.9 113.9 6.6 113.9 6.6 1136222.2 AD- 75.4 15.8 88.4 20.8 95.6 11.1 95.6 11.1 1297693.1 AD- 77.5 15.8 55.3 3.2 76.3 13.9 76.3 13.9 1297694.1 AD- 64.0 15.5 46.8 13.9 57.7 9.7 57.7 9.7 1136223.2 AD- 69.5 29.1 66.2 20.4 101.5 27.0 101.5 27.0 1136224.2 AD- 98.8 35.1 70.1 20.9 117.7 25.2 117.7 25.2 1136225.2 AD- 69.5 18.8 88.6 37.0 128.0 25.9 128.0 25.9 1297695.1 AD- 61.8 23.2 50.8 18.2 102.3 23.1 102.3 23.1 1297696.1 AD- 95.3 13.1 68.6 5.8 100.1 15.8 100.1 15.8 1297697.1 AD- 47.2 12.2 63.1 9.2 80.5 41.8 80.5 41.8 1136226.2 AD- 55.1 8.6 44.5 7.5 64.3 11.1 64.3 11.1 1136227.2 AD- 84.9 13.5 94.7 27.3 91.2 17.2 91.2 17.2 1136228.2 AD- 91.9 10.5 95.3 27.5 116.4 23.4 116.4 23.4 1136229.2 AD- 119.7 22.0 68.4 23.2 106.2 9.2 106.2 9.2 1136230.2 AD- 85.7 6.8 76.5 31.1 94.5 13.6 94.5 13.6 1136231.2 AD- 72.4 12.4 67.0 11.6 94.9 23.9 94.9 23.9 1136232.2 AD- 61.6 13.7 54.5 8.9 83.4 26.1 83.4 26.1 1297698.1 AD- 46.0 5.9 55.4 14.6 66.6 14.0 66.6 14.0 1297699.1 AD- 92.2 41.6 95.2 10.8 114.1 24.6 114.1 24.6 1297700.1 AD- 117.7 27.6 117.3 16.1 110.6 8.0 110.6 8.0 1136233.2 AD- 108.2 22.1 62.1 4.6 87.5 17.3 87.5 17.3 1136234.2 AD- 102.5 11.9 84.4 12.8 74.9 8.8 74.9 8.8 1297701.1 AD- 121.5 19.7 76.0 6.6 91.9 21.3 91.9 21.3 1297702.1 AD- 104.9 10.6 67.7 10.4 79.2 10.8 79.2 10.8 1297703.1 AD- 61.2 22.3 75.7 9.8 85.7 9.3 85.7 9.3 1297704.1 AD- 68.6 36.2 89.9 32.2 71.0 7.8 71.0 7.8 1297705.1 AD- 49.9 41.2 93.7 9.8 95.2 24.0 95.2 24.0 1297706.1 AD- 83.3 18.6 107.2 7.7 106.5 8.3 106.5 8.3 1297707.1 AD- 95.8 15.7 93.9 27.1 86.3 10.0 86.3 10.0 1297708.1 AD- 89.9 23.6 92.7 20.2 83.5 14.1 83.5 14.1 1297709.1 AD- 74.2 20.8 94.7 10.3 74.2 3.5 74.2 3.5 1297710.1 AD- 59.4 36.2 114.5 22.5 72.3 3.0 72.3 3.0 1297711.1 AD- 87.3 12.2 88.3 7.6 88.9 10.3 88.9 10.3 1297712.1

TABLE 10 Xanthine Dehydrogenase dsRNA Agent In Vitro Single Dose Screens in Primary Human Hepatocytes Dose (nM) 100 20 4 0.8 0.16 0.032 0.0064 0.00128 0.000256 5.12E−05 % Message AD-1395794.1 30.7 30.6 40.2 35.9 36.1 33.5 55.7 53.3 69.1 60.5 Remaining AD-1136038.3 43.5 36.9 40.5 35.5 43.0 59.8 61.4 61.1 94.7 76.9 Avg AD-1395797.1 45.8 45.8 53.6 68.5 58.9 70.2 100.1 89.7 128.6 105.1 AD-1135991.3 42.0 44.8 32.0 54.1 54.0 61.7 68.4 71.8 91.1 104.4 AD-1297597.2 56.7 51.3 46.3 43.4 56.9 76.7 86.5 89.4 75.7 84.5 AD-1395803.1 39.4 43.6 35.7 42.3 53.2 48.2 82.5 79.5 79.1 69.3 AD-1395805.1 32.7 45.1 34.5 43.1 43.3 81.0 84.0 76.3 85.7 100.7 PBS avg 106.5 Stdev AD-1395794.1 9 7 8 7 10 2 18 11 19 17 AD-1136038.3 11 11 8 9 5 10 16 16 19 10 AD-1395797.1 1 10 9 8 18 22 10 14 13 34 AD-1135991.3 13 14 15 15 17 10 18 18 22 35 AD-1297597.2 7 8 4 14 9 16 12 16 31 8 AD-1395803.1 10 5 4 15 18 17 14 28 16 19 AD-1395805.1 8 16 6 6 9 27 24 20 15 25

TABLE 11 Xanthine Dehydrogenase dsRNA Agent In Vitro Single Dose Screens in Primary Human Hepatocytes Dose (nM) 100 20 4 0.8 0.16 0.032 0.0064 0.00128 0.000256 5.12E−05 % Message AD-1395807.1 40.3 36.9 38.3 29.8 43.8 53.9 67.0 75.2 82.8 94.4 Remaining AD-1395811.1 45.7 42.5 39.9 47.9 64.4 64.7 80.8 99.3 114.2 98.0 Avg AD-1297663.2 45.6 37.8 36.3 44.2 48.4 57.6 60.0 73.9 92.3 85.7 AD-1136008.2 46.4 50.6 39.3 42.2 49.6 69.4 86.4 94.2 120.7 102.0 AD-1395816.1 49.1 56.9 54.4 52.0 47.0 60.6 79.4 115.4 96.7 97.6 AD-1136061.2 46.9 52.1 48.3 49.4 45.5 66.8 77.0 103.3 129.1 99.5 AD-1135987.2 43.4 35.6 34.5 38.2 35.6 52.6 67.4 84.2 88.2 89.4 PBS avg 100.3 Stdev AD-1395807.1 12 7 7 5 8 14 10 11 11 13 AD-1395811.1 9 9 9 7 9 3 11 21 13 20 AD-1297663.2 5 12 10 12 4 7 8 18 26 19 % Message AD-1395807.1 40.3 36.9 38.3 29.8 43.8 53.9 67.0 75.2 82.8 94.4 Remaining AD-1136008.2 2 12 9 9 7 9 18 24 27 22 Avg AD-1395816.1 6 1 7 7 5 11 6 29 17 30 AD-1136061.2 4 7 10 6 8 6 24 18 28 13 AD-1135987.2 4 8 8 6 6 14 23 12 8 23

TABLE 12 Xanthine Dehydrogenase dsRNA Agent In Vitro Single Dose Screens in Primary Human Hepatocytes Dose (nM) 100 20 4 0.8 0.16 0.032 0.0064 0.00128 0.000256 % Message AD-1395823.1 37.7 47.3 42.1 44.0 48.8 64.9 74.3 93.8 89.7 Remaining AD-1136166.3 47.2 50.7 42.3 49.4 64.4 77.1 86.3 98.9 102.1 Avg AD-1136169.3 48.0 49.2 38.7 45.8 61.7 67.0 64.8 81.2 111.5 PBS avg 100.5 Stdev AD-1395823.1 11 9 13 10 21 12 11 30 15 AD-1136166.3 15 14 14 12 12 11 8 23 14 AD-1136169.3 23 5 13 8 9 18 9 5 28

TABLE 13 IC₅₀ for Selected Xanthine Dehydrogenase dsRNA Agents Determined from In Vitro Single Dose Screens in Primary Human Hepatocytes Duplex ID IC50 (nM) AD-1395794.1 0.005 AD-1136038.3 0.057 AD-1395797.1 17.28 AD-1135991.3 0.369 AD-1297597.2 0.338 AD-1395803.1 0.127 AD-1395805.1 0.345 AD-1395807.1 0.055 AD-1395811.1 0.614 AD-1297663.2 0.087 AD-1136008.2 0.311 AD-1395816.1 0.25 AD-1136061.2 0.4 AD-1135987.2 0.032 AD-1395823.1 0.184 AD-1136166.3 1.149 AD-1136169.3 0.435

Example 4. In Vivo Screen in Non-Human Primates

Duplexes of interest identified from the in vitro studies were evaluated in vivo. In particular, the pharmacodynamic activity of various GalNAc-conjugated siRNAs targeting XDH was analyzed in Cynomolgus monkeys following a single subcutaneous injection of the siRNAs. Cynomolgus monkeys (3 females/group) in Groups 1 through 12 were subcutaneously administered vehicle (1×PBS) or a single 10 mg/kg dose of AD-1136091, AD-1395794, AD-1395805, AD-1136008, AD-1136038, AD-1395823, AD-1395816, AD-1136061, AD-1395811, or AD-1136166, or a single 5 mg/kg dose of AD-1135991. Group 13 was administered Allopurinol in vehicle of 0.5% carboxymethylcellulose sodium salt (prepared with sterile water for injection) at 10 mg/kg via daily oral gavage administration.

Liver samples were collected from Groups 1, 2, 5, 9, and 12 on Days −15, 21, 43 and Day 64. Liver samples were collected from Groups 3, 4, 6, 7, 8, 10, and 11 on Days −15, 21, and 42. Liver samples were collected from Group 13 on Day −15 and Day 15. All samples were analyzed for the levels of XDH messenger RNA (RT-qPCR) and XDH protein (Western blot).

For the RT-qPCR analysis, the mean liver XDH mRNA level relative to glycerol-3-phosphate dehydrogenase (GAPDH)] from each individual animal was normalized to its respective predose sample to determine the percentage of XDH mRNA relative to predose at each specified time point (FIG. 2).

Liver XDH protein levels were analyzed using western blot. The liver XDH protein level relative to beta-actin for each individual animal was normalized to its respective predose level to determine the percentage of XDH protein relative to predose at each specified time point (FIG. 3).

As shown in FIGS. 2 and 3, the greatest silencing for XDH mRNA was observed on Day 21 post dose with a maximal suppression of 66% observed for the AD-1136091 group. Partial recovery was observed on Days 43 and 64. A greater extent of silencing was observed for liver XDH protein with maximal suppression observed on Days 21 or 43 depending on the duplex tested. Up to a 91% reduction in XDH liver protein level was observed for animals treated with AD-1136091 with partial recovery seen by Day 64 post-dose. These results demonstrate that treatment with the exemplary duplex agents targeting XDH effectively reduced both the mRNA level and the protein level of XDH in vivo.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims. 

We claim:
 1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of xanthine dehydrogenase (XDH) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 19 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of nucleotides 2691-2722 of SEQ ID NO: 1, and the antisense strand comprises a nucleotide sequence selected from the group consisting of 5′-AAAUACTCUGAGAGAGAUCCUGG-3′(SEQ ID NO:485); 5′-AUAATACUCUGAGAGAGAUCCUG-3′(SEQ ID NO:486); 5′-AAUAAUACUCUGAGAGAGAUCCU-3′(SEQ ID NO:487); 5′-ACAUAAUACUCUGAGAGAGAUCC-3′(SEQ ID NO:488); 5′-ACCAUAAUACUCUGAGAGAGAUC-3′(SEQ ID NO:489); 5′-AUCCAUAAUACUCUGAGAGAGAU-3′(SEQ ID NO:490); 5′-AUUCCAUAAUACUCUGAGAGAGA-3′(SEQ ID NO:491); 5′-AGUUCCAUAAUACUCUGAGAGAG-3′(SEQ ID NO:492); 5′-ACGUUCCAUAAUACUCUGAGAGA-3′(SEQ ID NO:493); 5′-ACUCGUUCCAUAAUACUCUGAGA-3′(SEQ ID NO:494); and 5′-AGCUCGUUCCAUAAUACUCUGAG-3′(SEQ ID NO:495), wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus, and wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus and two phosphorothioate internucleotide linkages at the 3′-terminus.
 2. The dsRNA agent of claim 1, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxythimidine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′-phosphate mimic, a nucleotide comprising a 2′-phosphate, a thermally destabilizing nucleotide, a glycol modified nucleotide (GNA), and a 2-O—(N-methylacetamide) modified nucleotide; and combinations thereof.
 3. The dsRNA agent of claim 1, wherein the sense strand and the antisense strand are each independently 19-25 nucleotides in length.
 4. The dsRNA agent of claim 1, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide.
 5. The dsRNA agent of claim 1, further comprising a ligand.
 6. The dsRNA agent of claim 5, wherein the ligand is conjugated to the 3′ end of the sense strand of the dsRNA agent.
 7. The dsRNA agent of claim 6, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.
 8. The dsRNA agent of claim 7, wherein the ligand is


9. The dsRNA agent of claim 8, wherein the dsRNA agent is conjugated to the ligand as shown in the following schematic

and, wherein X is O or S.
 10. The dsRNA agent of claim 1, wherein the sense strand and the antisense strand comprise nucleotide sequences selected from the group consisting of 5′-AGGAUCUCUCUCAGAGUAUUU-3′(SEQ ID NO:126) and 5′-AAAUACTCUGAGAGAGAUCCUGG-3′(SEQ ID NO:485); 5′-GGAUCUCUCUCAGAGUAUUAU-3′(SEQ ID NO:127) and 5′-AUAATACUCUGAGAGAGAUCCUG-3′(SEQ ID NO:486); 5′-GAUCUCUCUCAGAGUAUUAUU-3′(SEQ ID NO:128) and 5′-AAUAAUACUCUGAGAGAGAUCCU-3′(SEQ ID NO:487); 5′-AUCUCUCUCAGAGUAUUAUGU-3′(SEQ ID NO:129) and 5′-ACAUAAUACUCUGAGAGAGAUCC-3′(SEQ ID NO:488); 5′-UCUCUCUCAGAGUAUUAUGGU-3′(SEQ ID NO:130) and 5′-ACCAUAAUACUCUGAGAGAGAUC-3′(SEQ ID NO:489); 5′-CUCUCUCAGAGUAUUAUGGAU-3′(SEQ ID NO:131) and 5′-AUCCAUAAUACUCUGAGAGAGAU-3′(SEQ ID NO:490); 5′-UCUCUCAGAGUAUUAUGGAAU-3′(SEQ ID NO:132) and 5′-AUUCCAUAAUACUCUGAGAGAGA-3′(SEQ ID NO:491); 5′-CUCUCAGAGUAUUAUGGAACU-3′(SEQ ID NO:133) and 5′-AGUUCCAUAAUACUCUGAGAGAG-3′(SEQ ID NO:492); 5′-UCUCAGAGUAUUAUGGAACGU-3′(SEQ ID NO:134) and 5′-ACGUUCCAUAAUACUCUGAGAGA-3′(SEQ ID NO:493); 5′-UCAGAGUAUUAUGGAACGAGU-3′(SEQ ID NO:135) and 5′-ACUCGUUCCAUAAUACUCUGAGA-3′(SEQ ID NO:494); and 5′-CAGAGUAUUAUGGAACGAGCU-3′(SEQ ID NO:136) and 5′-AGCUCGUUCCAUAAUACUCUGAG-3′(SEQ ID NO:495).
 11. The dsRNA agent of claim 10, wherein the sense strand and the antisense strand comprise nucleotide sequences selected from the group consisting of 5′-asgsgaucUfcUfCfUfcagaguauuu-3′(SEQ ID NO:844) and 5′-asAfsaudAc(Tgn)cugagaGfaGfauccusgsg-3′(SEQ ID NO:1203); 5′-gsgsaucuCfuCfUfCfagaguauuau-3′(SEQ ID NO:845) and 5′-asUfsaadTa(C2p)ucugagAfgAfgauccsusg-3 ‘(SEQ ID NO:1204); 5’-gsasucucUfcUfCfAfgaguauuauu-3′(SEQ ID NO:846) and 5′-asAfsuaaUfacucugaGfaGfagaucscsu-3 ‘(SEQ ID NO:1205); 5’-asuscucuCfuCfAfGfaguauuaugu-3′(SEQ ID NO:847) and 5′-asCfsauaAfuacucugAfgAfgagauscsc-3′(SEQ ID NO:1206); 5′-uscsucucUfcAfGfAfguauuauggu-3′(SEQ ID NO:848) and 5′-asCfscauAfauacucuGfaGfagagasusc-3 ‘(SEQ ID NO:1207); 5’-csuscucuCfaGfAfGfuauuauggau-3′(SEQ ID NO:849) and 5′-asUfsccaUfaauacucUfgAfgagagsasu-3′(SEQ ID NO:1208); 5′-uscsucucAfgAfGfUfauuauggaau-3′(SEQ ID NO:850) and 5′-asUfsuccAfuaauacuCfuGfagagasgsa-3′(SEQ ID NO:1209); 5′-csuscucaGfaGfUfAfuuauggaacu-3′(SEQ ID NO:851) and 5′-asGfsuudCc(Agn)uaauacUfcUfgagagsasg-3′(SEQ ID NO:1210); 5′-uscsucagAfgUfAfUfuauggaacgu-3′(SEQ ID NO:852); and 5′-asCfsguuCfcauaauaCfuCfugagasgsa-3′(SEQ ID NO:1211); 5′-uscsagagUfaUfUfAfuggaacgagu-3′(SEQ ID NO:853) and 5′-asCfsucgUfuccauaaUfaCfucugasgsa-3′(SEQ ID NO:1212); and 5′-csasgaguAfuUfAfUfggaacgagcu-3′(SEQ ID NO:854) and 5′-asGfscucGfuuccauaAfuAfcucugsasg-3′(SEQ ID NO:1213); wherein a, g, c, and u are 2′-O-methyl (2′-OMe) A, G, C, and U, respectively; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U, respectively; s is a phosphorothioate linkage; dA is 2′-deoxyadenosine-3′-phosphate; (Tgn) is a thymidine-glycol nucleic acid (GNA)S-Isomer; dT is 2′-deoxythimidine-3′-phosphate; (C2p) is cytidine-2′-phosphate; and (Agn) is an adenosine-glycol nucleic acid (GNA)S-Isomer.
 12. A pharmaceutical composition for inhibiting expression of a gene encoding xanthine dehydrogenase (XDH) comprising the dsRNA agent of claim
 1. 13. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of xanthine dehydrogenase (XDH) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises at least 19 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of nucleotides 2701-2721 of SEQ ID NO: 1, and the antisense strand comprises at least 19 contiguous nucleotides from the corresponding nucleotide sequence of SEQ ID NO: 2, wherein all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand are modified nucleotides, wherein the sense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus, and wherein the antisense strand comprises two phosphorothioate internucleotide linkages at the 5′-terminus and two phosphorothioate internucleotide linkages at the 3′-terminus.
 14. The dsRNA agent of claim 13, wherein at least one of the modified nucleotides is selected from the group consisting of a deoxy-nucleotide, a 3′-terminal deoxythimidine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a phosphorothioate group, a nucleotide comprising a methylphosphonate group, a nucleotide comprising a 5′-phosphate, a nucleotide comprising a 5′-phosphate mimic, a nucleotide comprising a 2′-phosphate, a thermally destabilizing nucleotide, a glycol modified nucleotide (GNA), and a 2-O—(N-methylacetamide) modified nucleotide; and combinations thereof.
 15. The dsRNA agent of claim 13, wherein the sense strand and the antisense strand are each independently 19-25 nucleotides in length.
 16. The dsRNA agent of claim 15, further comprising a ligand.
 17. The dsRNA agent of claim 16, wherein the ligand is conjugated to the 3′ end of the sense strand of the dsRNA agent.
 18. The dsRNA agent of claim 17, wherein the ligand is one or more GalNAc derivatives attached through a monovalent, bivalent, or trivalent branched linker.
 19. The dsRNA agent of claim 18, wherein the ligand is


20. The dsRNA agent of claim 19, wherein the dsRNA agent is conjugated to the ligand as shown in the following schematic

and, wherein X is O or S.
 21. The dsRNA agent of claim 13, wherein the antisense strand comprises the nucleotide sequence 5′-ACUCGUUCCAUAAUACUCUGAGA-3′(SEQ ID NO:494).
 22. The dsRNA agent of claim 21, wherein the sense strand comprises the nucleotide sequence 5′-UCAGAGUAUUAUGGAACGAGU-3′(SEQ ID NO:135) and the antisense strand comprise the nucleotide sequence 5′-ACUCGUUCCAUAAUACUCUGAGA-3′(SEQ ID NO:494).
 23. The dsRNA agent of claim 22, wherein the sense strand comprises the nucleotide sequence 5′-uscsagagUfaUfUfAfuggaacgagu-3′(SEQ ID NO:853) and the antisense strand comprises the nucleotide sequence 5′-asCfsucgUfuccauaaUfaCfucugasgsa-3′(SEQ ID NO:1212), wherein a, g, c, and u are 2′-O-methyl (2′-OMe) A, G, C, and U, respectively; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U, respectively; and s is a phosphorothioate linkage.
 24. The dsRNA agent of claim 23, further comprising a ligand.
 25. The dsRNA agent of claim 24, wherein the 3′-end of the sense strand is conjugated to a ligand as shown in the following schematic

wherein X is O.
 26. A pharmaceutical composition for inhibiting expression of a gene encoding xanthine dehydrogenase (XDH) comprising the dsRNA agent of claim
 13. 27. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of xanthine dehydrogenase (XDH) in a cell, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises the nucleotide sequence 5′-uscsagagUfaUfUfAfuggaacgagu-3′(SEQ ID NO:853) and the antisense strand comprises the nucleotide sequence 5′-asCfsucgUfuccauaaUfaCfucugasgsa-3′(SEQ ID NO:1212), wherein a, g, c, and u are 2′-O-methyl (2′-OMe) A, G, C, and U, respectively; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U, respectively; and s is a phosphorothioate linkage; and wherein the 3′-end of the sense strand is conjugated to a ligand as shown in the following schematic

wherein X is O.
 28. The dsRNA agent of claim 27, wherein the sense strand consists of the nucleotide sequence 5′-uscsagagUfaUfUfAfuggaacgagu-3′(SEQ ID NO:853) and the antisense strand consists of the nucleotide sequence 5′-asCfsucgUfuccauaaUfaCfucugasgsa-3′(SEQ ID NO:1212).
 29. A pharmaceutical composition for inhibiting expression of a gene encoding xanthine dehydrogenase (XDH) comprising the dsRNA agent of claim
 27. 